Methods of treating an inflammatory condition or sensitivity to an allergen with anti-IL-33 antibodies

ABSTRACT

The present invention provides antibodies that bind to interleukin-33 (IL-33) and methods of using the same. The invention includes antibodies that inhibit or attenuate IL-33-mediated signaling. The antibodies of the invention may function to block the interaction between IL-33 and ST2. Alternatively, certain antibodies of the invention inhibit or attenuate IL-33-mediated signaling without blocking the IL-33/ST2 interaction. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human IL-33 with high affinity. The antibodies of the invention are useful for the treatment of diseases and disorders associated with IL-33 signaling and/or IL-33 cellular expression, such as inflammatory diseases, or allergic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/205,512,filed Mar. 12, 2014, which claims the benefit under 35 USC § 119(e) ofU.S. Provisional Application No. 61/778,687, filed Mar. 13, 2013, and61/819,018, filed May 3, 2013, each of which is herein specificallyincorporated by reference in its entirety.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 1850U502-Sequence.txt,created on Aug. 26, 2016 and containing 110,978 bytes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for human IL-33, and methods ofuse thereof.

BACKGROUND

Interleukin-33 (IL-33) is a ligand for ST2, a toll-like/interleukin-1receptor super-family member that associates with an accessory protein,IL-1 RAcP (for reviews, see, e.g., Kakkar and Lee, Nature Reviews—DrugDiscovery 7(10):827-840 (2008), Schmitz et al., Immunity 23:479-490(2005); Liew et al., Nature Reviews—Immunology 10:103-110 (2010); US2010/0260770; US 2009/0041718). Upon activation of ST2/IL-1 RAcP byIL-33, a signaling cascade is triggered through downstream moleculessuch as MyD88 (myeloid differentiation factor 88) and TRAF6 (TNFreceptor associated factor 6), leading to activation of NFκB (nuclearfactor-κB), among others. IL-33 signaling has been implicated as afactor in a variety of diseases and disorders. (Liew et al., NatureReviews—Immunology 10:103-110 (2010)).

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind human interleukin-33(“IL-33”). The antibodies of the invention are useful, inter alia, forinhibiting IL-33-mediated signaling and for treating diseases anddisorders caused by or related to IL-33 activity and/or signaling.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

In one embodiment, the antibodies that bind specifically to humaninterleukin-33 are isolated fully human monoclonal antibodies.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibit orattenuate IL-33-mediated signaling.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof block theinteraction of IL-33 and ST2.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof block theinteraction of IL-33 and ST2 with an IC₅₀ value of less than about 10nM, or blocks greater than about 50% of the interaction of IL-33 and ST2as measured in an in vitro receptor/ligand binding assay at 25° C.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof do not block, oronly partially block the interaction of IL-33 and ST2.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof bind human IL-33with a binding dissociation equilibrium constant (K_(D)) of less thanabout 1 nM as measured in a surface plasmon resonance assay at 37° C.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof bind human IL-33with a dissociative half-life (t½) of greater than about 8 minutes asmeasured in a surface plasmon resonance assay at 37° C.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated degranulation of human basophils.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated degranulation of human basophils with an IC₅₀ of lessthan about 600 pM as measured in an in vitro basophil activation test(BAT).

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated IFN-gamma production from human PBMCs.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated IFN-gamma production from human PBMCs with an IC₅₀ ofless than about 25 nM as measured in an in vitro PBMC IFN-gammaproduction assay.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated IFN-gamma production from human PBMCs with an IC₅₀ ofless than about 3 nM as measured in an in vitro PBMC IFN-gammaproduction assay.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof inhibitIL-33-mediated IFN-gamma production from human PBMCs with an IC₅₀ ofless than about 0.5 nM as measured in an in vitro PBMC IFN-gammaproduction assay.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof reduce thefrequency of CD4+ T cells, eosinophils and ILC2 cells in the lungs whenadministered to an animal model of allergen-induced lung inflammation.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof reduces the levelof IL-4 and IL-5 in the lungs when administered to an animal model ofallergen-induced lung inflammation.

In one embodiment, the antibodies that bind specifically to humaninterleukin-33, or antigen-binding fragments thereof, when administeredto an animal model of allergen-induced lung inflammation, result in atleast a 4 fold reduction of IL-4 levels and/or at least a 5 foldreduction in IL-5 levels when compared to allergen-challenged animalsreceiving an isotype control antibody.

The present invention provides antibodies, or antigen-binding fragmentsthereof comprising a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258,274, 290, and 308, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, and 316, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,and 308/316.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,232, 248, 264, 280, 296, and 314, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; and a light chain CDR3 (LCDR3) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 16, 32,48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272,288, 304, and 322, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208,216/224, 232/240, 248/256, 264/272, 280/288, 296/304 and 314/322.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260,276, 292, and 310, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118,134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, and 312, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a light chain CDR1(LCDR1) domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156,172, 188, 204, 220, 236, 252, 268, 284, 300, and 318, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222,238, 254, 270, 286, 302, and 320, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H1M9559N);20-22-24-28-30-32 (e.g. H1M9566N); 36-38-40-44-46-48 (e.g. H1M9568N);52-54-56-60-62-64 (e.g. H4H9629P); 68-70-72-76-78-80 (e.g. H4H9633P);84-86-88-92-94-96 (e.g. H4H9640P); 100-102-104-108-110-112 (e.g.H4H9659P); 116-118-120-124-126-128 (e.g. H4H9660P);132-134-136-140-142-144 (e.g. H4H9662P); 148-150-152-156-158-160 (e.g.,H4H9663P); 164-166-168-172-174-176 (e.g. H4H9664P);180-182-184-188-190-192 (e.g., H4H9665P); 196-198-200-204-206-208 (e.g.H4H9666P); 212-214-216-220-222-224 (e.g. H4H9667P);228-230-232-236-238-240 (e.g. H4H9670P); 244-246-248-252-254-256 (e.g.H4H9671P); 260-262-264-268-270-272 (e.g. H4H9672P);276-278-280-284-286-288 (e.g. H4H9675P); 292-294-296-300-302-304 (e.g.H4H9676P); and 310-312-314-318-320-322 (H1M9565N).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds IL-33,wherein the antibody or fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266,274/282, 290/298, and 308/316. Methods and techniques for identifyingCDRs within HCVR and LCVR amino acid sequences are well known in the artand can be used to identify CDRs within the specified HCVR and/or LCVRamino acid sequences disclosed herein. Exemplary conventions that can beused to identify the boundaries of CDRs include, e.g., the Kabatdefinition, the Chothia definition, and the AbM definition. In generalterms, the Kabat definition is based on sequence variability, theChothia definition is based on the location of the structural loopregions, and the AbM definition is a compromise between the Kabat andChothia approaches. See, e.g., Kabat, “Sequences of Proteins ofImmunological Interest,” National Institutes of Health, Bethesda, Md.(1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martinet al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databasesare also available for identifying CDR sequences within an antibody.

In another aspect, the invention provides nucleic acid moleculesencoding anti-IL-33 antibodies or antigen-binding fragments thereof.Recombinant expression vectors carrying the nucleic acids of theinvention, and host cells into which such vectors have been introduced,are also encompassed by the invention, as are methods of producing theantibodies by culturing the host cells under conditions permittingproduction of the antibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, and 307, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofcomprising a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, 169, 185, 201, 217, 233, 249, 265, 281, 297, and 315, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247,263, 279, 295, and 313, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof;and a LCDR3 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143,159, 175, 191, 207, 223, 239, 255, 271, 287, 303, and 321, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofwhich further comprises a HCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83,99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, and 309,or a substantially identical sequence having at least 90%, at least 95%,at least 98%, or at least 99% homology thereof; a HCDR2 domain encodedby a nucleotide sequence selected from the group consisting of SEQ IDNO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229,245, 261, 277, 293, and 311, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%homology thereof; a LCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91,107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, and317, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205,221, 237, 253, 269, 285, 301, and 319, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof.

According to certain embodiments, the antibody or fragment thereofcomprises the heavy and light chain CDR sequences encoded by the nucleicacid sequences of SEQ ID NOs: 1 and 9 (e.g. H1M9559N), 17 and 25 (e.g.H1M9566N), 33 and 41 (e.g. H1M9568N), 49 and 57 (e.g. H4H9629P), 65 and73 (e.g. H4H9633P), 81 and 89 (e.g. H4H9640P), 97 and 105 (e.g.H4H9659P), 113 and 121 (e.g. H4H9660P), 129 and 137 (e.g. H4H9662P), 145and 153 (e.g. H4H9663P), 161 and 169 (e.g. H4H9664P), 177 and 185 (e.g.H4H9665P), 193 and 201 (e.g. H4H9666P), 209 and 217 (e.g. H4H9667P), 225and 233 (e.g. H4H9670P), 241 and 249 (e.g. H4H9671P), 257 and 265 (e.g.H4H9672P), 273 and 281 (e.g. H4H9675P), 289 and 297 (e.g. H4H9676P), or307 and 315 (H1M9565N).

The present invention includes anti-IL-33 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof, whichspecifically binds IL-33 and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-IL-33 antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-IL-33 antibody. Exemplary agentsthat may be advantageously combined with an anti-IL-33 antibody include,without limitation, other agents that inhibit IL-33 activity (includingother antibodies or antigen-binding fragments thereof, peptideinhibitors, small molecule antagonists, etc.) and/or agents, which donot directly bind IL-33 but nonetheless interfere with, block orattenuate IL-33-mediated signaling. In one embodiment the secondtherapeutic agent may be selected from the group consisting of anon-steroidal anti-inflammatory (NSAID), a corticosteroid, a bronchialdilator, an antihistamine, epinephrine, a decongestant, a thymic stromallymphopoietin (TSLP) antagonist, an IL-13 antagonist, an IL-4antagonist, an IL-5 antagonist, an IL-6 antagonist, an IL-25 antagonist,an IL-17 antagonist, and another IL-33 antagonist or a differentantibody to IL-33. In certain embodiments, the cytokine antagonist maybe a small molecule inhibitor (synthetic or naturally derived), or aprotein (e.g. an antibody) that interacts with either the cytokineitself, or to a receptor for the cytokine, or to a complex comprisingboth the cytokine and its receptor(s) (e.g. an antibody to IL-4 or IL-6,or an antibody to the receptor for IL-4 or IL-6). Additional combinationtherapies and co-formulations involving the anti-IL-33 antibodies of thepresent invention are disclosed elsewhere herein.

In yet another aspect, the invention provides therapeutic methods forinhibiting IL-33 activity using an anti-IL-33 antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody orantigen-binding fragment of an antibody of the invention. The disordertreated is any disease or condition which is improved, ameliorated,inhibited or prevented by removal, inhibition or reduction of IL-33activity or signaling. The anti-IL-33 antibodies or antibody fragmentsof the invention may function to block the interaction between IL-33 andan IL-33 binding partner (e.g., an IL-33 receptor component), orotherwise inhibit the signaling activity of IL-33.

In one embodiment, the invention provides a method for treating aninflammatory disease or disorder, or at least one symptom associatedwith the inflammatory disease or disorder, the method comprisingadministering an antibody that binds specifically to IL-33, or anantigen-binding fragment thereof, or a pharmaceutical compositioncomprising an antibody that binds specifically to IL-33, or anantigen-binding fragment thereof, to a patient in need thereof, whereinthe inflammatory disease or disorder is alleviated, or reduced inseverity, duration or frequency of occurrence, or at least one symptomassociated with the inflammatory disease or disorder is alleviated, orreduced in severity, duration, or frequency of occurrence.

In one embodiment, the inflammatory disease or condition is selectedfrom the group consisting of asthma, atopic dermatitis, chronicobstructive pulmonary disease (COPD), inflammatory bowel disease,multiple sclerosis, arthritis, allergic rhinitis, eosinophilicesophagitis and psoriasis.

In one embodiment, the invention provides a method for treating apatient who demonstrates a sensitivity to an allergen, the methodcomprising administering an effective amount of an antibody or antigenbinding fragment thereof that binds specifically to IL-33, or apharmaceutical composition comprising an antibody that bindsspecifically to IL-33, or an antigen-binding fragment thereof, to apatient in need thereof, wherein the patient demonstrates a reducedsensitivity to, or a diminished allergic reaction against the allergen,or does not experience any sensitivity or allergic reaction to, oranaphylactic response to the allergen following administration of theantibody or a composition comprising the antibody.

In one embodiment, the invention provides for administering an effectiveamount of a second therapeutic agent useful for alleviating theinflammatory disease or disorder, or at least one symptom of theinflammatory disease or disorder, or for diminishing an allergicresponse to an allergen. As noted above, the second therapeutic agentmay be selected from the group consisting of a non-steroidalanti-inflammatory (NSAID), a corticosteroid, a bronchial dilator, anantihistamine, epinephrine, a decongestant, a thymic stromallymphopoietin (TSLP) antagonist, an IL-13 antagonist, an IL-4antagonist, an IL-4/IL-13 dual antagonist, an IL-5 antagonist, an IL-6antagonist, an IL-12/23 antagonist, an IL-22 antagonist, an IL-25antagonist, an IL-17 antagonist, an IL-31 antagonist, an oral PDE4inhibitor and another IL-33 antagonist or a different antibody to IL-33.

In a related aspect, the invention provides an anti-IL-33 antibody ofthe invention, or an antigen-binding fragment thereof, or apharmaceutical composition comprising the antibody or antigen-bindingfragment thereof for use in treating a disease or disorder related to,or caused by IL-33 activity in a patient. In one embodiment, the diseaseor disorder related to, or caused by IL-33 activity in a patient is aninflammatory disease or disorder, wherein the inflammatory disease ordisorder is selected from the group consisting of asthma, atopicdermatitis, chronic obstructive pulmonary disease (COPD), inflammatorybowel disease, multiple sclerosis, arthritis, allergic rhinitis,eosinophilic esophagitis and psoriasis.

The present invention also includes the use of an anti-IL-33 antibody orantigen binding portion of an antibody of the invention in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by IL-33 activity in a patient. In one embodiment,the disease or disorder related to, or caused by IL-33 activity in apatient is an inflammatory disease or disorder, wherein the inflammatorydisease or disorder is selected from the group consisting of asthma,atopic dermatitis, chronic obstructive pulmonary disease (COPD),inflammatory bowel disease, multiple sclerosis, arthritis, allergicrhinitis, eosinophilic esophagitis and psoriasis.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Cross Competition between Anti-IL-33 Antibodies for Human IL-33

FIG. 2. Cross Competition between Anti-IL-33 Antibodies for RecombinantMonkey IL-33

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expressions “interleukin-33,” “IL-33,” and the like, as used herein,refer to a human IL-33 protein as obtained from, for example, R&DSystems, catalogue #3625-IL-010/CF. All references to proteins,polypeptides and protein fragments herein are intended to refer to thehuman version of the respective protein, polypeptide or protein fragmentunless explicitly specified as being from a non-human species (e.g.,“mouse IL-33,” “monkey IL-33,” etc.).

As used herein, “an antibody that binds IL-33” or an “anti-IL-33antibody” includes antibodies, and antigen-binding fragments thereof,that bind a soluble fragment of an IL-33 protein. Soluble IL-33molecules include natural IL-33 proteins as well as recombinant IL-33protein variants such as, e.g., monomeric and dimeric IL-33 constructs.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., IL-33). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-IL-33 antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-IL-33 antibodies ofthe invention are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention includes neutralizing and/or blocking anti-IL-33antibodies. A “neutralizing” or “blocking” antibody, as used herein, isintended to refer to an antibody whose binding to IL-33: (i) interfereswith the interaction between IL-33 or an IL-33 fragment and an IL-33receptor component (e.g., ST2, IL-1 RAcP, etc.); and/or (ii) results ininhibition of at least one biological function of IL-33. The inhibitioncaused by an IL-33 neutralizing or blocking antibody need not becomplete so long as it is detectable using an appropriate assay.Exemplary assays for detecting IL-33 inhibition are described in theworking Examples herein.

The anti-IL-33 antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-IL-33 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-IL-33 antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

An “inflammatory disease or disorder”, as used herein, refers to adisease, disorder or pathological condition where the pathology results,in whole or in part, from, e.g., a change in number, change in rate ofmigration, or change in activation, of cells of the immune system. Cellsof the immune system include, e.g., T cells, B cells, monocytes ormacrophages, antigen presenting cells (APCs), dendritic cells,microglia, NK cells, neutrophils, eosinophils, mast cells, or any othercell specifically associated with the immunology, for example,cytokine-producing endothelial or epithelial cells. As used herein, inone embodiment, the “inflammatory disease or disorder” is an immunedisorder or condition selected from the group consisting of asthma,(including steroid resistant asthma, steroid sensitive asthma,eosinophilic asthma or non-eosinophilic asthma, allergy, anaphylaxis,multiple sclerosis, inflammatory bowel disorder (e.g. Crohn's disease orulcerative colitis), chronic obstructive pulmonary disease (COPD, whichmay or may not be related to, caused in part by, or resulting from,exposure to first or second hand cigarette smoke), lupus, atopicdermatitis, psoriasis, scleroderma and other fibrotic diseases,sjogren's syndrome, vasculitis (behcet's disease, Giant cell arteritis,Henoch-Schonlein purpura and Churg Strauss syndrome) and arthritis. Inanother embodiment, the arthritis is selected from the group consistingof rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. Inanother embodiment, the “inflammatory disease or disorder” is an immunedisorder or condition comprises a TH₁-type response or a TH₂-typeresponse.

The phrase “Inhibits or attenuates IL-33-mediated signaling”, as usedherein, refers to the degree to which IL-33 stimulates signaltransduction through ST2 and IL-1 RAcP, which is diminished in thepresence of an antagonist, such as an IL-33 antibody as describedherein, relative to the degree to which IL-33 stimulates signaltransduction through ST2 and IL-1 RAcP in the absence of the antagonistsuch as an IL-33 antibody as described herein. To examine the extent ofinhibition, a sample is treated with a potential inhibitor/antagonistand is compared to a control sample without the inhibitor/antagonist.Control samples, i.e., not treated with antagonist, are assigned arelative activity value of 100%. Inhibition is achieved when theactivity value relative to the control is about 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less. An endpointin inhibition may comprise a predetermined quantity or percentage of,e.g., an indicator of inflammation, or cell degranulation, secretion oractivation, such as the release of a cytokine. Inhibition of IL-33signal transduction through ST2 and IL-1 RAcP can be determined byassaying for IL-33 signal transduction in an in vitro assay, such asthat described herein in Example 6. In addition, in vivo assays can beused to determine whether a molecule is an antagonist of IL-33. Forexample, an in vivo assay such as that described in Examples 11 and 12may be used to assess the effect of an antibody to IL-33 on lunginflammation in allergen-sensitized animals that are homozygous forexpression of human IL-33. Following sensitization of the animals withallergen, a subset of the animals is treated with either an anti-IL-33antibody of the invention or a negative isotype control antibody.Afterwards, the animals are sacrificed and the lungs are harvested forassessment of cellular infiltrates, as well as cytokine measurements(IL-4 and IL-5). An IL-33 antibody that is effective as an antagonistshould demonstrate a trend towards reduction in inflammatory cells inthe lung, as well as a trend towards reduction in cytokines such as IL-4and IL-5.

Biological Characteristics of the Antibodies

The present invention includes anti-IL-33 antibodies and antigen-bindingfragments thereof that bind human IL-33 and inhibit or attenuateIL-33-mediated signaling. An anti-IL-33 antibody is deemed to “inhibitor attenuate IL-33-mediated signaling” if, e.g., the antibody exhibitsone or more properties selected from the group consisting of: (1)inhibition of IL-33-mediated signaling in a cell-based bioassay; (2)inhibition of IL-33-induced degranulation of human basophils; (3)inhibition of IL-33-induced IFNγ production from human PBMCs; (4)reduction in cytokine levels that are elevated in a mammal as a resultof exposure to an allergen, e.g. IL-4 or IL-5; and (5) inhibition oflung inflammation resulting from acute or chronic exposure to anallergen (e.g. house dust mites (HDM)).

Inhibition of IL-33-mediated signaling in a cell-based bioassay meansthat an anti-IL-33 antibody or antigen-binding fragment thereof inhibitsor reduces the signal produced in cells that express an IL-33 receptorand a reporter element that produces a detectable signal in response toIL-33 binding, e.g., using the assay format as defined in Example 6herein, or a substantially similar assay. For example, the presentinvention includes antibodies and antigen-binding fragments thereof thatblock IL-33-mediated signaling in cells expressing human ST2, with anIC₅₀ of less than about 2 nM, less than about 1 nM, less than about 900pM, less than about 800 pM, less than about 700 pM, less than about 600pM, less than about 500 pM, less than about 400 pM, less than about 350pM, less than about 300 pM, less than about 250 pM, less than about 200pM, less than about 150 pM, less than about 100 pM, less than about 90pM, less than about 80 pM, less than about 70 pM, less than about 60 pM,less than about 50 pM, less than about 40 pM, less than about 30 pM,less than about 20 pM, or less than about 10 pM, as measured in acell-based blocking bioassay, e.g., using the assay format as defined inExample 5 herein, or a substantially similar assay.

Inhibition of IL-33-induced degranulation of human basophils means thatan anti-IL-33 antibody or antigen-binding fragment thereof inhibits orreduces the extent of IL-33-induced basophil degranulation in vitro,e.g., as measured using the assay system of Example 7 or a substantiallysimilar assay. For example, the present invention includes antibodiesand antigen-binding fragments thereof that inhibit degranulation ofhuman basophils in the presence of human IL-33 (e.g., about 100 pM finalconcentration), with an IC₅₀ of less than about 500 pM, less than about400 pM, less than about 350 pM, less than about 300 pM, less than about250 pM, less than about 200 pM, less than about 150 pM, less than about100 pM, less than about 90 pM, less than about 80 pM, less than about 70pM, less than about 60 pM, less than about 50 pM, less than about 40 pM,less than about 30 pM, less than about 20 pM, or less than about 10 pM,as measured in an in vitro human basophil degranulation assay, e.g.,using the assay format as defined in Example 7 herein, or asubstantially similar assay.

Inhibition of IL-33-induced IFNγ production from human PBMCs means thatan anti-IL-33 antibody or antigen-binding fragment thereof inhibits orreduces the amount of IFNγ released from PBMCs treated with human IL-33in the presence of human IL-12, e.g., as measured using the assay systemof Example 8 or a substantially similar assay. For example, the presentinvention includes antibodies and antigen-binding fragments thereof thatinhibit IL-33-induced release of IFNγ, in the presence of human IL-12,with an IC₅₀ of less than about 50 nM, less than about 25 nM, less thanabout 20 nM, less than about 15 nM, less than about 10 nM, less thanabout 5 nM, less than about 1 nM, less than about 900 pM, less thanabout 800 pM, less than about 700 pM, less than about 600 pM, less thanabout 500 pM, less than about 400 pM or less than about 300 pM, asmeasured in an IL-33-induced IFNγ release assay, e.g., using the assayformat as defined in Example 8 herein, or a substantially similar assay.

In certain embodiments, the anti-IL-33 antibodies and antigen-bindingfragments of the present invention block the binding of IL-33 to anIL-33 receptor (e.g., ST2). For example, the present invention includesanti-IL-33 antibodies that block the binding of IL-33 to ST2 in vitro,with an IC₅₀ value of less than about 15 nM, as measured by anELISA-based immunoassay, e.g., using the assay format as defined inExample 4 herein, or a substantially similar assay. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention block the binding of IL-33 to ST2 in vitro with an IC₅₀ valueof less than about 10 nM, less than about 5 nM, less than about 900 pM,less than about 800 pM, less than about 700 pM, less than about 600 pM,less than about 500 pM, less than about 400 pM, less than about 300 pM,less than about 280 pM, less than about 260 pM, less than about 250 pM,less than about 240 pM, less than about 230 pM, less than about 220 pM,less than about 200 pM, less than about 180 pM, less than about 160 pM,or less than about 150 pM, as measured by an ELISA-based immunoassay,e.g., using the assay format as defined in Example 4 herein, or asubstantially similar assay.

In other embodiments, however, certain anti-IL-33 antibodies andantigen-binding fragments of the present invention, despite having theability to inhibit or attenuate IL-33-mediated signaling, do not blockor only partially block the interaction of IL-33 and ST2. Suchantibodies and antigen-binding fragments thereof, may be referred toherein as “indirect blockers.” Without being bound by theory, it isbelieved that the indirect blockers of the invention function by bindingto IL-33 at an epitope that does overlap, or overlaps only partially,with the ST2-binding domain of IL-33, but nonetheless interfere withIL-33-mediated signaling without blocking the IL-33/ST2 interactiondirectly.

The present invention includes anti-IL-33 antibodies and antigen-bindingfragments thereof that bind soluble IL-33 molecules with high affinity.For example, the present invention includes antibodies andantigen-binding fragments of antibodies that bind IL-33 (e.g., at 25° C.or 37° C.) with a K_(D) of less than about 10 nM as measured by surfaceplasmon resonance, e.g., using the assay format as defined in Example 3herein. In certain embodiments, the antibodies or antigen-bindingfragments of the present invention bind IL-33 with a K_(D) of less thanabout 5 nM, less than about 2 nM, less than about 1 nM, less than about800 pM, less than about 600 pM, less than about 500 pM, less than about400 pM, less than about 300 pM, less than about 200 pM, less than about180 pM, or less than about 160 pM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes anti-IL-33 antibodies andantigen-binding fragments thereof that specifically bind to IL-33 with adissociative half-life (t½) of greater than about 10 minutes as measuredby surface plasmon resonance at 25° C. or 37° C., e.g., using the assayformat as defined in Example 3 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind IL-33 with a t½ of greater than about 20minutes, greater than about 30 minutes, greater than about 40 minutes,greater than about 50 minutes, greater than about 60 minutes, greaterthan about 70 minutes, greater than about 80 minutes, greater than about90 minutes, greater than about 100 minutes, as measured by surfaceplasmon resonance at 25° C. or 37° C., e.g., using the assay format asdefined in Example 3 herein, or a substantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Anti-IL-33 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-IL-33antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-IL-33 antibodies comprising a mutationin the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/For Y); or a modification at position 250 and/or 428; or a modificationat position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment,the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P). In yet another embodiment, themodification comprises a 265A (e.g., D265A) and/or a 297A (e.g., D297A)modification.

For example, the present invention includes anti-IL-33 antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433Kand N434F). All possible combinations of the foregoing Fc domainmutations, and other mutations within the antibody variable domainsdisclosed herein, are contemplated within the scope of the presentinvention.

The present invention also includes anti-IL-33 antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Provisional Appl. No.61/759,578, filed Feb. 1, 2013, the disclosure of which is herebyincorporated by reference in its entirety).

Epitope Mapping and Related Technologies

The present invention includes anti-IL-33 antibodies which interact withone or more amino acids of IL-33. For example, the present inventionincludes anti-IL-33 antibodies that interact with one or more aminoacids located within the ST2-interacting domain of IL-33. The epitope towhich the antibodies bind may consist of a single contiguous sequence of3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) amino acids of IL-33. Alternatively, the epitope mayconsist of a plurality of non-contiguous amino acids (or amino acidsequences) of IL-33.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-IL-33 antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. H1M9559N, H1M9566N, H1M9568N, H4H9629P, H4H9633P,H4H9640P, H4H9659P, H4H9660P, H4H9662P, H4H9663P, H4H9664P, H4H9665P,H4H9666P, H4H9667P, H4H9670P, H4H9671P, H4H9672P, H4H9675P, H4H9676P,H1M9565N, etc.). Likewise, the present invention also includesanti-IL-33 antibodies that compete for binding to IL-33 with any of thespecific exemplary antibodies described herein (e.g. H1M9559N, H1M9566N,H1M9568N, H4H9629P, H4H9633P, H4H9640P, H4H9659P, H4H9660P, H4H9662P,H4H9663P, H4H9664P, H4H9665P, H4H9666P, H4H9667P, H4H9670P, H4H9671P,H4H9672P, H4H9675P, H4H9676P, H1M9565N, etc.).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-IL-33 antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-IL-33 antibody of the invention, the reference antibodyis allowed to bind to an IL-33 protein. Next, the ability of a testantibody to bind to the IL-33 molecule is assessed. If the test antibodyis able to bind to IL-33 following saturation binding with the referenceanti-IL-33 antibody, it can be concluded that the test antibody binds toa different epitope than the reference anti-IL-33 antibody. On the otherhand, if the test antibody is not able to bind to the IL-33 moleculefollowing saturation binding with the reference anti-IL-33 antibody,then the test antibody may bind to the same epitope as the epitope boundby the reference anti-IL-33 antibody of the invention. Additionalroutine experimentation (e.g., peptide mutation and binding analyses)can then be carried out to confirm whether the observed lack of bindingof the test antibody is in fact due to binding to the same epitope asthe reference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, Biacore, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present invention, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20- or 100-fold excess of one antibody inhibits binding of theother by at least 50% but preferably 75%, 90% or even 99% as measured ina competitive binding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-IL-33 antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to an IL-33protein under saturating conditions followed by assessment of binding ofthe test antibody to the IL-33 molecule. In a second orientation, thetest antibody is allowed to bind to an IL-33 molecule under saturatingconditions followed by assessment of binding of the reference antibodyto the IL-33 molecule. If, in both orientations, only the first(saturating) antibody is capable of binding to the IL-33 molecule, thenit is concluded that the test antibody and the reference antibodycompete for binding to IL-33. As will be appreciated by a person ofordinary skill in the art, an antibody that competes for binding with areference antibody may not necessarily bind to the same epitope as thereference antibody, but may sterically block binding of the referenceantibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human IL-33.

Using VELOCIMMUNE™ technology, for example, or any other known methodfor generating fully human monoclonal antibodies, high affinity chimericantibodies to IL-33 are initially isolated having a human variableregion and a mouse constant region. As in the experimental sectionbelow, the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Ifnecessary, mouse constant regions are replaced with a desired humanconstant region, for example wild-type or modified IgG1 or IgG4, togenerate a fully human anti-IL-33 antibody. While the constant regionselected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region. In certain instances, fully human anti-IL-33 antibodiesare isolated directly from antigen-positive B cells.

Bioequivalents

The anti-IL-33 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman IL-33. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-IL-33 antibody-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-IL-33 antibody or antibody fragment that isessentially bioequivalent to an anti-IL-33 antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-IL-33 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-IL-33 antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-IL-33 antibodies that bind to human IL-33 but not to IL-33 fromother species. The present invention also includes anti-IL-33 antibodiesthat bind to human IL-33 and to IL-33 from one or more non-humanspecies. For example, the anti-IL-33 antibodies of the invention maybind to human IL-33 and may bind or not bind, as the case may be, to oneor more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog,rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesusor chimpanzee IL-33. According to certain exemplary embodiments of thepresent invention, anti-IL-33 antibodies are provided which specificallybind human IL-33 and cynomolgus monkey (e.g., Macaca fascicularis)IL-33.

Immunoconjugates

The invention encompasses anti-IL-33 monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-IL-33 antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human IL-33 or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

pH-Dependent Binding

The present invention provides antibodies and antigen-binding fragmentsthereof that bind IL-33 in a pH-dependent manner. For example, ananti-IL-33 antibody of the invention may exhibit reduced binding toIL-33 at acidic pH as compared to neutral pH. Alternatively, ananti-IL-33 antibody of the invention may exhibit enhanced binding to itsantigen at acidic pH as compared to neutral pH.

In certain instances, “reduced binding to IL-33 at acidic pH as comparedto neutral pH” is expressed in terms of a ratio of the K_(D) value ofthe antibody binding to IL-33 at acidic pH to the K_(D) value of theantibody binding to IL-33 at neutral pH (or vice versa). For example, anantibody or antigen-binding fragment thereof may be regarded asexhibiting “reduced binding to IL-33 at acidic pH as compared to neutralpH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained. As used herein, the expression “acidic pH” means a pH of about6.0 or less, about 5.5 or less, or about 5.0 or less. The expression“acidic pH” includes pH values of about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75,5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1,5.05, 5.0, or less. As used herein, the expression “neutral pH” means apH of about 7.0 to about 7.4. The expression “neutral pH” includes pHvalues of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-IL-33 antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with IL-33 activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-IL-33antibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

Experiments using mouse model systems, conducted by the presentinventors, have contributed to the identification of various diseasesand conditions that can be treated, prevented and/or ameliorated byIL-33 antagonism. For example, hydrodynamic delivery of mouse IL-33 DNAresulted in the induction of lung mucus accumulation and increases intotal serum IgE in mice. In addition, mIL-33 DNA delivery resulted inup-regulation of ST2 and various downstream cytokines as measured bymicroarray analysis. Experiments conducted by the present inventorsusing IL-33 knock-out mice also revealed various potential therapeuticbenefits of IL-33 antagonism. For example, macroscopic scoring and skininfiltrates were found to be comparable between wild-type mice andIL-33^(−/−) mice in a model of IMQ-induced psoriasis. Moreover,IL-33^(−/−) mice showed reduced eosinophilia and residual mucusaccumulation in an allergen-induced lung inflammation model.

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by IL-33 expression, signaling, or activity,or treatable by blocking the interaction between IL-33 and a IL-33ligand (e.g., ST2) or otherwise inhibiting IL-33 activity and/orsignaling. For example, the present invention provides methods fortreating, asthma (e.g., allergic asthma, non-allergic asthma, severerefractory asthma, asthma exacerbations, steroid resistant asthma,steroid sensitive asthma, eosinophilic asthma or non-eosinophilicasthma, etc.), atopic dermatitis, psoriasis, other inflammatorydisorders, allergy, anaphylaxis, cardiovascular disease, central nervoussystem disease, pain, arthritis (e.g., rheumatoid arthritis,osteoarthritis, psoriatic arthritis, etc.), giant cell arteritis,vasculitis (behcet's disease and Churg Strauss syndrome),Henoch-Schonlein purpura., multiple sclerosis, inflammatory boweldisorder (e.g. Crohn's disease or ulcerative colitis), lupus, andsjogren's syndrome.

The antibodies of the present invention are also useful for thetreatment, prevention and/or amelioration of one or more fibroticdiseases. Exemplary fibrotic diseases that are treatable byadministering the anti-IL-33 antibodies of the invention includepulmonary fibrosis (e.g., idiopathic pulmonary fibrosis,bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonaryfibrosis, and bronchiolitis obliterans syndrome), chronic asthma,fibrosis associated with acute lung injury and acute respiratorydistress (e.g., bacterial pneumonia induced fibrosis, trauma inducedfibrosis, viral pneumonia induced fibrosis, ventilator induced fibrosis,non-pulmonary sepsis induced fibrosis and aspiration induced fibrosis),silicosis, radiation-induced fibrosis, chronic obstructive pulmonarydisease (COPD, which may or may not be related to, caused in part by, orresulting from, exposure to first or second hand cigarette smoke),scleroderma, ocular fibrosis, skin fibrosis (e.g., scleroderma), hepaticfibrosis (e.g., cirrhosis, alcohol-induced liver fibrosis, non-alcoholicsteatohepatitis (NASH), bilary duct injury, primary bilary cirrhosis,infection- or viral-induced liver fibrosis, autoimmune hepatitis, kidney(renal) fibrosis, cardiac fibrosis, atherosclerosis, stent restenosis,and myelofibrosis.

In the context of the methods of treatment described herein, theanti-IL-33 antibody may be administered as a monotherapy (i.e., as theonly therapeutic agent) or in combination with one or more additionaltherapeutic agents (examples of which are described elsewhere herein).

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the anti-IL-33 antibodies described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof.

The anti-IL-33 antibodies of the present invention may be co-formulatedwith and/or administered in combination with, e.g., cytokine inhibitors,including small-molecule cytokine inhibitors and antibodies that bind tocytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11,IL-12, IL-13, IL-17, IL-18, IL-21, IL-23, IL-25, IL-26, or antagonistsof their respective receptors.

The anti-IL-33 antibodies of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids, steroids, oxygen, antioxidants, metalchelators, IFN-gamma, and/or NSAIDs.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan anti-IL-33 antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-IL-33 antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-IL-33 antibody ofthe present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-IL-33 antibody (or a pharmaceutical compositioncomprising a combination of an anti-IL-33 antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-IL-33 antibody ofthe invention. As used herein, “sequentially administering” means thateach dose of anti-IL-33 antibody is administered to the subject at adifferent point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of an anti-IL-33 antibody, followed byone or more secondary doses of the anti-IL-33 antibody, and optionallyfollowed by one or more tertiary doses of the anti-IL-33 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-IL-33 antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-IL-33 antibody, but generally may differ from one another in termsof frequency of administration. In certain embodiments, however, theamount of anti-IL-33 antibody contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-IL-33 antibody which is administeredto a patient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-IL-33 antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.).

Diagnostic Uses of the Antibodies

The anti-IL-33 antibodies of the present invention may also be used todetect and/or measure IL-33, or IL-33-expressing cells in a sample,e.g., for diagnostic purposes. For example, an anti-IL-33 antibody, orfragment thereof, may be used to diagnose a condition or diseasecharacterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of IL-33. Exemplarydiagnostic assays for IL-33 may comprise, e.g., contacting a sample,obtained from a patient, with an anti-IL-33 antibody of the invention,wherein the anti-IL-33 antibody is labeled with a detectable label orreporter molecule. Alternatively, an unlabeled anti-IL-33 antibody canbe used in diagnostic applications in combination with a secondaryantibody which is itself detectably labeled. The detectable label orreporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I; a fluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure IL-33 in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in IL-33 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of IL-33 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of IL-33 in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal IL-33 levels or activity) will be measured to initiallyestablish a baseline, or standard, level of IL-33. This baseline levelof IL-33 can then be compared against the levels of IL-33 measured insamples obtained from individuals suspected of having a IL-33 relateddisease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human IL-33

An immunogen comprising human IL-33 was administered directly, with anadjuvant to stimulate the immune response, to a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The antibody immune response was monitored by anIL-33-specific immunoassay. When a desired immune response was achievedsplenocytes were harvested and fused with mouse myeloma cells topreserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce IL-33-specific antibodies. Using this technique severalanti-IL-33 chimeric antibodies (i.e., antibodies possessing humanvariable domains and mouse constant domains) were obtained; exemplaryantibodies generated in this manner were designated as follows:H1M9559N, H1M9566N, H1M9568N and H1M9565N. The human variable domainsfrom the chimeric antibodies were subsequently cloned onto humanconstant domains to make fully human anti-IL-33 antibodies as describedherein.

Anti-IL-33 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in US2007/0280945A1. Using this method, several fully human anti-IL-33antibodies (i.e., antibodies possessing human variable domains and humanconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as follows: H4H9629P, H4H9633P, H4H6940P,H4H9659P, H4H9660P, H4H9662P, H4H9663P, H4H9664P, H4H9665P, H4H9666P,H4H9667P, H4H9670P, H4H9671P, H4H9672P, H4H9675P, and H4H9676P.

Certain biological properties of the exemplary anti-IL-33 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs, and CDR sequences, of selected anti-IL-33 antibodies andtheir corresponding antibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 9559N 2 4 6 8 10 12 14 16 9566N 18 20 22 24 26 28 3032 9568N 34 36 38 40 42 44 46 48 9629P 50 52 54 56 58 60 62 64 9633P 6668 70 72 74 76 78 80 9640P 82 84 86 88 90 92 94 96 9659P 98 100 102 104106 108 110 112 9660P 114 116 118 120 122 124 126 128 9662P 130 132 134136 138 140 142 144 9663P 146 148 150 152 154 156 158 160 9664P 162 164166 168 170 172 174 176 9665P 178 180 182 184 186 188 190 192 9666P 194196 198 200 202 204 206 208 9667P 210 212 214 216 218 220 222 224 9670P226 228 230 232 234 236 238 240 9671P 242 244 246 248 250 252 254 2569672P 258 260 262 264 266 268 270 272 9675P 274 276 278 280 282 284 286288 9676P 290 292 294 296 298 300 302 304 9565N 308 310 312 314 316 318320 322

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” or “H4H”), followed by a numericalidentifier (e.g. “9559,” “9566,” or “9629” as shown in Table 1),followed by a “P,” or “N” suffix. Thus, according to this nomenclature,an antibody may be referred to herein as, e.g., “H1M9559N,” “H1M9566N,”“H4H9629P,” etc. The H1M and H4H prefixes on the antibody designationsused herein indicate the particular Fc region isotype of the antibody.For example, an “H1M” antibody has a mouse IgG1 Fc, whereas an “H4H”antibody has a human IgG4 Fc. As will be appreciated by a person ofordinary skill in the art, an antibody having a particular Fc isotypecan be converted to an antibody with a different Fc isotype (e.g., anantibody with a mouse IgG1 Fc can be converted to an antibody with ahuman IgG4, etc.), but in any event, the variable domains (including theCDRs)—which are indicated by the numerical identifiers shown in Table1—will remain the same, and the binding properties are expected to beidentical or substantially similar regardless of the nature of the Fcdomain.

Example 3 Antibody Binding to Human IL-33 as Determined by SurfacePlasmon Resonance

Equilibrium dissociation constants (K_(D) values) for IL-33 binding topurified anti-IL-33 monoclonal antibodies were determined using areal-time surface plasmon resonance biosensor using a Biacore 4000instrument. The Biacore sensor surface was first derivatized by aminecoupling with either a polyclonal rabbit anti-mouse antibody (GE, #BR-1008-38) or with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture anti-IL-33 monoclonal antibodies expressed withmouse or with human IgG4 constant regions, respectively. All Biacorebinding studies were performed in 0.01M ADA pH 7.4, 0.15M NaCl, 3 mMEDTA, and 0.05% v/v Surfactant Tween-20 (ABS-ET running buffer).Different concentrations of human IL-33 (hIL-33; R&D Systems,#3625-IL-010/CF) or cynomolgus monkey IL-33 expressed with a C-terminalhexahistidine tag (MfIL-33-6His; SEQ ID NO: 305) prepared in ABS-ETrunning buffer (ranging from 100 nM to 3.7 nM, 3-fold dilutions) wereinjected over the anti-IL-33 monoclonal antibody captured surface at aflow rate of 30 μ/minute. Association of either hIL-33 or MfIL-33-6Histo the captured monoclonal antibody was monitored for 4 minutes andtheir dissociation in ABS-ET running buffer was monitored for 10minutes. The effect of reduced pH on the binding of each anti-IL-33antibody to either hIL-33 or MfIL-33-6His was studied using an in-linepH chase assay format in 0.01M ADA pH 6.0, 0.15M NaCl, 3 mM EDTA, and0.05% v/v Surfactant Tween-20 (ABS-ET pH6 buffer). To achieve this,association of either hIL-33 or MfIL-33-6His to the captured monoclonalantibody was monitored for 4 minutes in ABS-ET running buffer. Followinga 30 second dissociation of either hIL-33 or MfIL-33-6His in ABS-ETrunning buffer, ABS-ET pH6 buffer was injected for 3 minutes, and theanalyte dissociation under the low-pH conditions was measured. All thebinding kinetic experiments were performed at both 25° C. and 37° C.Kinetic association (k_(a)) and dissociation (k_(d)) constants weredetermined by fitting the real-time sensorgrams to a 1:1 binding modelusing Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as:K _(D)(M)=k _(d) /k _(a) and t _(1/2) (min)=ln(2)/(60*k _(d))

Binding kinetic parameters for hIL-33 and MfIL-33-6His binding todifferent anti-IL-33 monoclonal antibodies at 25° C. and 37° C. areshown in Tables 2 through 5. At 25° C., hIL-33bound to the anti-IL-33antibodies with K_(D) values ranging from 78 pM to 757 pM, as shown inTable 2. At 37° C., hIL-33 bound to the anti-IL-33 antibodies with K_(D)values ranging from 411 pM to 2.03 nM, as shown in Table 3. At both 25°C. and 37° C., one anti-IL-33 antibody demonstrated weak binding andtherefore its binding kinetic parameters could not be fit using an 1:1binding model. At 25° C., MfIL-33-6His bound to the anti-IL-33antibodies with K_(D) values ranging from 333 pM to 38 nM, as shown inTable 4. At 37° C., MfIL-33-6His bound to the anti-IL-33 antibodies withK_(D) values ranging from 1 nM to 48.6 nM, as shown in Table 5.

TABLE 2 Binding kinetic parameters of anti-IL-33 monoclonal antibodiesbinding to human IL-33 at 25° C. In-Line Chase in ABS-ET pH 6 BufferHuman IL-33 Binding Kinetics t½ in ABS-ET Running Buffer Ratio Antibodyk_(a) k_(d) K_(D) t½ k_(d) t½ (pH 7.4/ Captured (1/Ms) (1/s) (M) (min)(1/s) (min) pH 6.0) H4H9675P 1.02E+06 2.58E−04 2.54E−10 45 1.11E−03 104.3 H4H9662P 8.11E+05 2.50E−04 3.08E−10 46 8.26E−04 14 3.3 H4H9640P9.12E+05 2.37E−04 2.60E−10 49 6.57E−04 18 2.8 H4H9629P 7.77E+05 2.26E−042.90E−10 51 1.28E−03 9 5.7 H4H9659P 5.26E+05 1.72E−04 3.27E−10 676.64E−04 17 3.9 H4H9660P 6.96E+05 2.24E−04 3.22E−10 52 7.08E−04 16 3.2H4H9667P 6.37E+05 2.52E−04 3.95E−10 46 5.66E−04 20 2.2 H4H9670P 7.86E+052.89E−04 3.68E−10 40 8.25E−04 14 2.9 H4H9663P 1.36E+06 4.14E−04 3.05E−1028 1.10E−03 11 2.7 H4H9666P 5.08E+05 2.80E−04 5.51E−10 41 1.34E−03 9 4.8H4H9676P 1.03E+06 3.45E−04 3.34E−10 33 1.21E−03 10 3.5 H4H9633P 6.56E+052.83E−04 4.32E−10 41 8.10E−04 14 2.9 H4H9671P 7.71E+05 3.49E−04 4.53E−1033 1.62E−03 7 4.6 H4H9672P 6.68E+05 3.52E−04 5.27E−10 33 1.41E−03 8 4.0H4H9665P 8.88E+05 4.74E−04 5.33E−10 24 2.12E−03 5 4.5 H4H9664P 3.39E+052.57E−04 7.57E−10 45 8.23E−04 14 3.2 H1M9568N 7.02E+05 1.30E−04 1.84E−1089 1.78E−04 65 1.4 H1M9566N 1.27E+05  1.00E−05**  7.88E−11** 1155**1.10E−04 105 11.0 H1M9559N 4.04E+05 2.74E−04 6.78E−10 42 1.87E−04 62 0.7H1M9565N IC* IC* IC* IC* IC* IC* IC* *IC: inconclusive since very weakbinding was observed under the experimental conditions and the real-timebinding data could not be reliably fit into the 1:1 binding model.**Under the experimental conditions no dissociation of IL33 from thecaptured monoclonal antibody was observed' therefore the value of k_(d)was fixed to 1.00E−05, and the derived t½ and K_(D) values representlower and upper limits, respectively.

TABLE 3 Binding kinetic parameters of anti-IL-33 monoclonal antibodiesbinding to human IL-33 at 37° C. In-Line Chase in ABS-ET pH 6 BufferHuman IL-33 Binding Kinetics t½ in ABS-ET Running Buffer Ratio Antibodyk_(a) k_(d) K_(D) t½ k_(d) t½ (pH 7.4/ Captured (1/Ms) (1/s) (M) (min)(1/s) (min) pH 6.0) H4H9675P 2.12E+06 8.72E−04 4.11E−10 13 4.63E−03 25.3 H4H9662P 1.40E+06 6.20E−04 4.43E−10 19 3.83E−03 3 6.2 H4H9640P1.15E+06 5.73E−04 4.98E−10 20 2.65E−03 4 4.6 H4H9629P 1.27E+06 6.46E−045.08E−10 18 5.82E−03 2 9.0 H4H9659P 7.07E+05 4.03E−04 5.70E−10 292.99E−03 4 7.4 H4H9660P 8.03E+05 4.79E−04 5.96E−10 24 3.23E−03 4 6.8H4H9667P 9.76E+05 6.03E−04 6.18E−10 19 2.44E−03 5 4.0 H4H9670P 1.16E+067.83E−04 6.76E−10 15 3.83E−03 3 4.9 H4H9663P 1.83E+06 1.24E−03 6.77E−109 4.62E−03 3 3.7 H4H9666P 1.13E+06 7.70E−04 6.81E−10 15 6.80E−03 2 8.8H4H9676P 1.38E+06 1.28E−03 9.22E−10 9 5.24E−03 2 4.1 H4H9633P 7.40E+056.89E−04 9.31E−10 17 2.40E−03 5 3.5 H4H9671P 1.21E+06 1.14E−03 9.38E−1010 5.85E−03 2 5.1 H4H9672P 1.09E+06 1.15E−03 1.05E−09 10 5.41E−03 2 4.7H4H9665P 1.21E+06 1.44E−03 1.19E−09 8 9.65E−03 1 6.7 H4H9664P 5.19E+057.21E−04 1.39E−09 16 2.79E−03 4 3.9 H1M9568N 6.72E+05 9.61E−04 1.43E−0912 1.10E−03 10 1.1 H1M9566N 1.66E+05 2.83E−04 1.70E−09 41 9.67E−04 123.4 H1M9559N 4.73E+05 9.62E−04 2.03E−09 12 9.92E−04 12 1.0 H1M9565N IC*IC* IC* IC* IC* IC* IC* *IC: inconclusive since very weak binding wasobserved under the experimental conditions and the real-time bindingdata could not be reliably fit into the 1:1 binding model.

TABLE 4 Binding kinetic parameters of anti-IL-33 monoclonal antibodiesbinding to MfIL-33-6His at 25° C. In-Line Chase in ABS-ET pH 6 BufferMfIL-33-6His Binding Kinetics t½ in ABS-ET Running Buffer Ratio Antibodyk_(a) k_(d) K_(D) t½ k_(d) t½ (pH 7.4/ Captured (1/Ms) (1/s) (M) (min)(1/s) (min) pH 6.0) H4H9675P 5.06E+05 1.29E−03 2.55E−09 9 1.56E−03 7 1.2H4H9662P 3.53E+05 4.42E−04 1.25E−09 26 1.17E−04 99 0.3 H4H9640P 4.50E+051.37E−03 3.06E−09 8 5.01E−04 23 0.4 H4H9629P 5.62E+05 1.35E−02 2.39E−080.9 3.58E−02 0.3 2.7 H4H9659P 3.25E+05 4.86E−04 1.50E−09 24 1.23E−04 940.3 H4H9660P 4.26E+05 1.49E−03 3.49E−09 8 1.08E−03 11 0.7 H4H9667P3.43E+05 9.91E−04 2.89E−09 12 6.96E−04 17 0.7 H4H9670P 4.40E+05 2.10E−034.77E−09 6 3.93E−04 29 0.2 H4H9663P 8.69E+05 9.25E−04 1.06E−09 126.83E−04 17 0.7 H4H9666P 2.22E+05 3.54E−03 1.59E−08 3.3 8.09E−03 1.4 2.3H4H9676P 8.52E+05 4.12E−03 4.84E−09 2.8 1.45E−03 8 0.4 H4H9633P 2.62E+059.97E−03 3.80E−08 1.2 2.87E−03 4 0.3 H4H9671P 5.87E+05 1.50E−03 2.55E−098 1.61E−03 7 1.1 H4H9672P 4.37E+05 3.60E−03 8.22E−09 3.2 2.67E−03 4 0.7H4H9665P 5.57E+05 5.66E−04 1.02E−09 20 7.53E−04 15 1.3 H4H9664P 1.40E+051.65E−03 1.18E−08 7 4.80E−04 24 0.3 H1M9568N 2.44E+05 2.61E−04 1.07E−0944 3.02E−04 38 1.2 H1M9566N 2.93E+05 9.75E−05 3.33E−10 119 1.26E−04 911.3 H1M9559N 3.21E+05 1.23E−03 3.82E−09 9 1.52E−03 8 1.2 H1M9565N4.06E+04 7.20E−05 1.77E−09 160 1.43E−04 81 2.0

TABLE 5 Binding kinetic parameters of anti-IL-33 monoclonal antibodiesbinding to MfIL-33-6His at 37° C. In-Line Chase in ABS-ET pH 6 BufferMfIL-33-6His Binding Kinetics t½ in ABS-ET Running Buffer Ratio Antibodyk_(a) k_(d) K_(D) t½ k_(d) t½ (pH 7.4/ Captured (1/Ms) (1/s) (M) (min)(1/s) (min) pH 6.0) H4H9675P 1.02E+06 4.91E−03 4.81E−09 2.4 7.35E−03 1.61.5 H4H9662P 7.07E+05 1.58E−03 2.24E−09 7 1.68E−03 7 1.1 H4H9640P8.10E+05 4.36E−03 5.38E−09 2.6 2.26E−03 5 0.5 H4H9629P 1.07E+06 3.47E−023.24E−08 0.3 FT* FT* FT* H4H9659P 5.98E+05 1.86E−03 3.11E−09 6 1.02E−0311 0.5 H4H9660P 6.80E+05 4.44E−03 6.53E−09 2.6 4.63E−03 2.5 1.0 H4H9667P6.81E+05 3.17E−03 4.66E−09 4 2.68E−03 4 0.8 H4H9670P 7.35E+05 5.03E−036.84E−09 2.3 1.65E−03 7 0.3 H4H9663P 1.62E+06 3.61E−03 2.22E−09 3.23.54E−03 3.3 1.0 H4H9666P 4.32E+05 1.41E−02 3.27E−08 0.8 FT* FT* FT*H4H9676P 1.87E+06 1.44E−02 7.70E−09 0.8 FT* FT* FT* H4H9633P 4.68E+052.27E−02 4.86E−08 0.5 FT* FT* FT* H4H9671P 1.20E+06 6.07E−03 5.08E−091.9 8.19E−03 1.4 1.3 H4H9672P 9.46E+05 1.30E−02 1.37E−08 0.9 FT* FT* FT*H4H9665P 1.10E+06 2.10E−03 1.91E−09 5 4.00E−03 2.9 1.9 H4H9664P 3.61E+055.84E−03 1.62E−08 2.0 1.93E−03 6 0.3 H1M9568N 3.89E+05 1.73E−03 4.46E−097 2.24E−03 5 1.3 H1M9566N 3.99E+05 4.00E−04 1.00E−09 29 1.15E−03 10 2.9H1M9559N 4.93E+05 3.47E−03 7.04E−09 3.3 3.07E−03 4 0.9 H1M9565N 7.82E+042.02E−04 2.59E−09 57 2.28E−04 51 1.1 *FT: fast t½.

Example 4 Anti-IL-33 Antibodies Block Binding of IL-33 to the Human ST2Receptor

The ability of anti-IL-33 antibodies to block either human IL-33(hIL-33) or cynomologus monkey IL-33 binding to the human ST2 receptorwas measured using a competition sandwich ELISA. A portion of human ST2protein ecto domain that was expressed with a C-terminal human IgG1 Fctag (hST2-hFc; SEQ ID NO:306), was coated at a concentration of 1 μg/mLin PBS buffer on a 96-well microtiter plate overnight at 4° C.Nonspecific binding sites were subsequently blocked using a 0.5% (w/v)solution of BSA in PBS. Constant concentrations of either 30 pMbiotinylated hIL-33 protein (R&D systems, Cat #3625-IL/CF)(biotin-hIL-33) or 150 pM cynomologus monkey IL-33 expressed withhexahistidine tag (MfIL-33-6His; SEQ ID NO:305) were separately added toserial dilutions of antibodies so that the final concentrations ofantibodies ranged from 0 to 100 nM. The antibody/IL-33 mixtures wereincubated for 1 hour at room temperature before they were transferred tothe hST2-hFc-coated microtiter plates. After incubating for 1 hour atroom temperature, the wells were then washed, and plate-boundbiotin-hIL-33 was detected with streptavidin conjugated withhorse-radish peroxidase (HRP) (Thermo Scientific, Cat # N200), andplate-bound MfIL-33-6His was detected with a HRP conjugated anti-Hismonoclonal antibody (Qiagen, #34460). All samples were developed with aTMB solution (BD biosciences, #51-2607KC) to produce a colorimetricreaction and then quenched by acidification with 1M sulfuric acid beforemeasuring absorbance at 450 nm on a Victor X5 plate reader. Dataanalysis was performed using a sigmoidal dose-response model withinPrism™ software. The calculated IC₅₀ value, defined as the concentrationof antibody required to reduce by 50% from maximal signal thebiotin-hIL-33 or MfIL-33-6His binding to plate-coated hST2-hFc, was usedas an indicator of blocking potency. Percent blockade was calculated asthe ratio of the reduction in signal observed in the presence ofantibody relative to the difference between the signal with IL-33 aloneand background (signal from HRP-conjugated secondary antibody orstreptavidin alone). The absorbance measured for the constantconcentration of biotin-hIL-33 or MfIL-33-6His alone is defined as 0%blocking and the absorbance measured for no added IL-33 is defined as100% blocking. The absorbance values of the wells containing the highestconcentration for each antibody were used to determine the percentmaximum blocking.

TABLE 6 ELISA blocking of biotin-hIL-33 or MfIL-33-6His binding tohST2-hFc by anti-IL-33 antibodies Blocking 30 pM biotin- % MaximumBlocking 150 pM % Maximum hIL-33 on blocking biotin- Mf-IL-33-6Hisblocking Mf-IL- hST2-hFc, hIL-33 on on hST2-hFc, 33-6His on Ab ID IC₅₀(M) hST2-hFc IC₅₀ (M) hST2-hFc H1M9559N* 1.4E−10 88 1.0E−08 53 H1M9566N*3.2E−10 69 2.2E−10 41 H1M9565N* 2.2E−08 68 1.2E−08 86 H1M9568N* 1.9E−1055 8.4E−10 38 H4H9629P 4.5E−10 80 N/A NBI H4H9633P 4.4E−10 66 N/A NBIH4H9640P 3.5E−10 78 3.5E−09 73 H4H9659P 4.0E−10 78 6.0E−10 92 H4H9660P3.1E−10 57 4.2E−09 68 H4H9662P 1.0E−09 77 8.6E−10 87 H4H9663P 5.0E−10 741.2E−09 81 H4H9664P 3.0E−10 73 3.8E−09 67 H4H9665P 8.7E−10 55 4.2E−10 81H4H9666P 6.0E−10 71 1.3E−08 40 H4H9667P 4.1E−10 78 4.1E−09 72 H4H9670P4.8E−10 69 3.5E−09 69 H4H9671P 4.6E−10 46 5.8E−10 62 H4H9672P 4.4E−10 635.5E−09 48 H4H9675P 4.4E−10 58 1.5E−09 72 H4H9676P 4.6E−10 54 3.2E−09 57N/A = not applicable NBI = non-blocker *= Experiment performed on aseparate day

Binding experiments for 20 antibodies were performed on two separatedays, as indicated in Table 6. All 20 of the anti-IL-33 antibodiesblocked biotin-hIL-33 binding to hST2-hFc with IC₅₀ values ranging from140 pM to 22 nM and percent maximum blocking ranging from 46% to 88%.Eighteen of the 20 anti-IL-33 antibodies blocked MfIL-33-6His binding tohST2-hFc with IC₅₀ values ranging from 220 pM to 13 nM and percentmaximum blocking ranging from 38% to 92%, as shown in Table 6. Two ofthe antibodies tested, H4H9629P and H4H9633P, did not demonstratemeasurable blockade of MfIL-33-6His binding to hST2-hFc.

Example 5 Inhibition of IL-33 Binding to Anti-IL-33 Monoclonal Antibodyby ST2 as Shown by Biacore Analysis

The ability of anti-IL-33 antibodies to bind to a pre-formed complex ofIL-33 with ST2 was tested using Biacore T-200 instrument equipped with areal-time surface plasmon resonance biosensor. The experiment wasperformed at 25° C. with a running buffer composed of 0.01M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, and 0.05% v/v Surfactant Tween-20 (HBS-ET).The Biacore sensor surface was first derivatized by amine coupling ananti-myc tag-specific monoclonal antibody (Clone#9E10), and on thisderivatized sensor was captured approximately 160 response units (RU) ofhuman ST2 protein expressed with a C-terminal myc-myc-hexahistidine tag(hST2-MMH; SEQ ID NO: 323). The captured hST2-MMH surface was thensaturated by injecting 100 nM of human IL-33 (hIL-33; R&D Systems,#3625-IL-010/CF) for 3 minutes followed by a 3 minute injection of a 100nM solution of the anti-IL-33 monoclonal antibody. The real-time bindingresponse was monitored during the entire course of the experiment, andthe observed binding response at 3 minutes after injection of anti-IL-33antibody to the pre-formed complex of hIL-33 and captured hST2-MMH wasrecorded and tabulated and shown in Table 7. No non-specific binding ofanti-IL-33 monoclonal antibody to the anti-myc tag capture surface wasobserved. As shown in Table 7, 17 of the tested antibodies did not showmeasurable binding to hIL-33 after it was pre-complexed with hST2-MMH,while three antibodies (H1M9565N, H1M9566N, and H1M9568N) bound tohIL-33 after it was pre-complexed with hST2-MMH.

TABLE 7 Binding of anti-IL-33 antibodies to a pre-formed complex ofhIL-33 and hST2-MMH Antibody Binding Antibody Response (RU) H4H9629P −1H4H9633P −1 H4H9640P −1 H4H9659P −1 H4H9660P −1 H4H9662P 0 H4H9663P −1H4H9664P −1 H4H9665P 0 H4H9666P −1 H4H9667P −1 H4H9670P −1 H4H9671P −1H4H9672P −1 H4H9675P −1 H4H9676P −1 H1M9559N −4 H1M9565N 11 H1M9566N 13H1M9568N 131

Example 6 Inhibition of IL-33-Mediated Receptor Signaling by Anti-IL-33Antibodies

Interleukin-33 (IL-33) is a ligand for ST2, a toll-like/interleukin-1receptor super-family member that associates with an accessory protein,IL-1 RAcP (for review, see Kakkar and Lee, 2008). Upon activation ofST2/IL-1 RAcP by IL-33, a signaling cascade is triggered throughdownstream molecules such as MyD88 (myeloid differentiation factor 88)and TRAF6 (TNF receptor associated factor 6), leading to activation ofNFκB (nuclear factor—κB), among others. To develop a biologicallyrelevant bioassay system to test anti-IL-33 antibodies, human embryonickidney cells (HEK293) were stably transfected to express human ST2(amino acids 1-556 of accession number NP_057316) along with aluciferase reporter [NFκB response element (5×)-luciferase-IRES-GFP](HEK293/hST2/NFkB-luciferase cell line). The HEK293 cell line expressesIL-1 RAcP endogenously and NFκB activation by IL-33 in HEK293 cells hasbeen shown previously (Schmitz et al., Immunity 23:479-490 (2005)). Thestable cell line was isolated and maintained in 10% FBS, DMEM, NEAA,penicillin/streptomycin, and G418.

For the bioassay, HEK293/hST2/NFkB-luciferase cells were seeded onto96-well assay plates at 10,000 cells per well in low serum mediacontaining 0.1% w/v FBS and OPTIMEM (Invitrogen, #31985-070) and thenincubated at 37° C. in 5% CO₂ overnight. The next day, to determine thedose response of IL-33, either human IL-33 (hIL-33; R&D Systems,#3625-IL) or cynomolgus monkey IL-33 expressed with a C-terminalhexahistidine tag (MfIL-33-6His; SEQ ID N0:305) were serially diluted at1:3 and added to the cells starting from 10 nM and ranging down to0.0002 nM, plus a control sample containing no IL-33. To measureinhibition, antibodies were serially diluted and added to the cellsfollowed by addition of constant concentrations of IL-33 (10 pM hIL-33for the human assay and 5 pM MfIL-33-6His for the monkey assay).Three-fold antibody serial dilutions were performed before adding to thecells, starting from 100 pM and ranging down to 0.002 nM or startingfrom 10 nM and ranging down to 0.0002 nM. In addition to the antibodydilution series, a well containing the constant concentration of IL-33but without any antibody was also included. After 5.5 hours ofincubation at 37° C. in 5% CO₂, luciferase activity was detected using aVictor X (Perkin Elmer) plate reader, and the results were analyzedusing nonlinear regression (4-parameter logistics) with Prism 5. Resultsare shown in Table 8.

TABLE 8 Inhibition of human IL-33 and monkey IL-33 activation ofHEK293/hST2/NFkB-luciferase cells by anti-IL33 antibodies Species HumanMonkey EC₅₀ [M] 2.2E−12 3.5E−12 2.4E−11 8.2E−13 3.5E−12 Constant IL-3310 pM hIL-33 5 pM MfIL-33-6His AbPID IC₅₀ [M] Notes IC₅₀ [M] IC₅₀ [M]IC₅₀ [M] Notes IC₅₀ [M] H1M9559N 2.0E−09 4.9E−08 H1M9566N 9.5E−10Partial 1.5E−09 Partial Inhibition Inhibition (Max at 66%) (Max at 61%)H1M9565N 2.9E−08 1.7E−08 H1M9568N 2.5E−10 Partial 3.5E−09 PartialInhibition Inhibition (Max at 48%) (Max at 34%) H4H9629P 1.3E−11 5.5E−08H4H9633P 2.2E−10 1.3E−07 H4H9640P 3.0E−11 1.4E−08 H4H9659P 4.7E−113.3E−09 H4H9660P 3.5E−11 1.9E−08 H4H9662P 2.0E−11 1.5E−09 H4H9663P1.3E−10 2.7E−09 H4H9664P 5.0E−11 2.6E−08 H4H9665P 9.0E−11 6.6E−10H4H9666P 3.5E−11 7.8E−08 H4H9667P 7.1E−11 1.2E−08 H4H9670P 1.2E−101.7E−08 H4H9671P 2.5E−11 4.8E−09 H4H9672P 2.5E−11 2.0E−08 H4H9675P7.5E−12 4.1E−09 H4H9676P 3.5E−11 8.4E−09

Eighteen of the 20 anti-IL33 antibodies blocked human IL-33 stimulationof the HEK293/hST2/NFkB-luciferase cells with IC₅₀ values ranging from7.5 pM to 29 nM, as shown in Table 8. Two of the antibodies tested,H1M9566N and H1M9568N, partially inhibited hIL-33 with a maximuminhibition of 48% and 66%, with IC₅₀ values of 950 pM and 250 pM,respectively. Eighteen of the 20 anti-IL33 antibodies blockedMfIL-33-6His stimulation of HEK293/hST2/NFkB-luciferase cells with IC₅₀values ranging from 660 pM to 130 nM as shown in Table 8. Two of theantibodies tested, H1M9566N and H1M9568N, partially inhibitedMfIL-33-6His with a maximum inhibition of 61% and 34%, with IC₅₀ valuesof 1.5 nM and 3.5 nM, respectively.

Example 7 Inhibition of IL-33-Induced Degranulation of Human Basophilsby Anti-IL-33 Antibodies

To further assess the in vitro characteristics of select anti-IL-33antibodies of the invention, their ability to block IL-33-inducedbasophil degranulation was measured. Peripheral blood mononuclear cells(PBMC) were purified from fresh whole blood from two different humandonors by density gradient centrifugation. K2 EDTA whole blood wasdiluted 1:1 in RPMI 1640, carefully layered over Ficoll-Paque (GEHealthcare, #17-1440-03) and centrifuged to separate PBMC. Theinterphase layer containing the PBMC was aspirated, transferred to a newtube, and washed twice with MACS buffer that was comprised of a 1:20dilution of the MACS BSA solution (Militenyi Biotec, #130-091-376) inMACS rinsing solution (Militenyi Biotec, #130-091-222). The purifiedPBMC were then plated in a v-bottom 96-well plate at a finalconcentration of ˜3.0×10⁶ cells/mL in 100 μL of MACS buffer. To primethe basophils contained within the PBMC population, 1 ng of IL-3 (Sigma,# H7166-10UG) in 50 μL Dulbecco's Phosphate-Buffered Saline without Ca⁺⁺or Mg⁺⁺ (DPBS) was added to the cell suspension, and then incubated at37° C. for 10 minutes.

Serial dilutions (1:3) of two different exemplary anti-IL-33 antibodiesof the invention (H4H9675P and H4H9659P) or an isotype control antibodywere made, ranging from 10 nM to 4.6 pM, plus a control with noantibody. The solutions were mixed with a fixed concentration of 100 pM(final concentration) of human IL-33 (R&D Systems, #3625-IL/CF) or noIL-33 negative control prior to adding to the PBMC. All conditions weretested in duplicate.

After addition of the human IL-33 and antibodies to the cells, the cellswere incubated at 37° C. for 20 minutes to facilitate basophildegranulation. Degranulation was then stopped by cooling the assayplates on wet ice for 5 minutes. To enable analysis of the basophilpopulation used to measure degranulation, 20 μL each (as per themanufacturer's instructions) of anti-HLA-DR-FITC (Beckman Coulter, #IM0463U), anti-CD123-APC (BD, #560087), and anti-CD203c-PE (BeckmanCoulter, # IM3575) were added to each sample, and the samples were heldat 4° C. for 20 minutes in the dark. The cells were then centrifuged,washed with DPBS, and then resuspended in 2% formaldehyde (fixationbuffer) at 4° C. The next day, fixed cells were analyzed on a BDFACSCanto II to determine levels of basophil degranulation. Results aresummarized in Tables 9 and 10.

TABLE 9 Percent degranulation of human basophils induced by human IL-33challenge 100 pM IL-33 No IL-33 Donor Mean SD Mean SD 655687 68.8002.263 10.295 0.856 655688 61.600 0.849 9.915 0.969

TABLE 10 Anti-IL-33 antibody blocking human IL-33 induced degranulationof human basophils Donor 655687 Donor 655688 Antibody IC₅₀ (M) IC₅₀ (M)H4H9675P 1.329E−10 9.712E−11 H4H9659P 5.786E−10 4.465E−10 IsotypeControl non-blocking non-blocking

As shown in Table 9, at 100 pM, human IL-33 induced basophildegranulation in two different donors with a mean percent degranulationof 68.8% for donor 655687 and 61.6% for donor 655688.

As shown in Table 10, one anti-IL33 antibody, H4H9675P, blocked basophildegranulation induced by 100 pM human IL-33 challenge with an IC₅₀ valueof 132.9 pM for donor 655687, and an IC₅₀ value of 97.12 pM for donor655688. Another anti-IL33 antibody, H4H9659P, blocked basophildegranulation induced by 100 pM human IL-33 challenge with an IC₅₀ valueof 578.6 pM for donor 655687, and an IC₅₀ value of 446.5 pM for donor655688. In contrast, the isotype control did not block basophildegranulation from any of the tested donors.

Example 8 Inhibition of IL-33-Induced IFN-gamma from Human PBMCs byAnti-IL-33 Antibodies

To further characterize anti-IL-33 antibodies of the invention, aprimary cell based assay using peripheral blood mononuclear cells(PBMCs) was utilized. The assay used in this Example was based on theresults published by Smithgall et al. in International Immunology, 2008,vol. 20 (8) pp. 1019-1030. For this assay, PBMCs were purified fromfresh whole blood from three different donors by density gradientcentrifugation. Briefly, K2 EDTA whole blood was diluted two-fold inRPMI 1640, carefully layered over Ficoll-Paque (GE Healthcare,#17-1440-03) and centrifuged for 20 minutes. The interphase layercontaining the PBMCs was aspirated, transferred to a new tube, andwashed twice with PBS. The isolated PBMCs were plated in round-bottom96-well plates at a final concentration of 5×10⁵ cells/mL in RPMI 1640supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and100 μg/mL streptomycin. Cells were then incubated with 50 g/mL of humanIL-12 (hIL-12; R&D Systems, #219-IL-025/CF) and a serial dilution ofhuman IL-33 (hIL-33; R&D Systems, #3625-IL-010/CF) alone from 10 nM to0.64 pM, or with 260 pM of hIL-33 in combination with serial dilutionsof antibodies from 100 nM to 6.4 pM. The final volume was 200 μL perwell. Each condition was tested in triplicate. When antibodies werepresent, they were added to the cells after 30 minutes of pre-incubationwith hIL-33.

The cells were incubated overnight at 37° C. in a humidified incubatorwith 5% CO₂, and then IFNγ levels in the culture supernatant weremeasured by ELISA (R&D Systems, #DY285). For the ELISA, 96-wellflat-bottom plates were coated with the capture antibody, according tothe manufacturer's instructions. After washing and blocking, 100 μL ofundiluted culture supernatant was added to the plates and incubated for2 hours. Subsequent washes and detection were done following themanufacturer's instructions.

Human IL-33, in the presence of hIL-12, induced the release of IFNγ fromhuman total PBMC from the three different donors tested, with EC₅₀values between 274 pM to 39 pM as shown in Table 11. Eleven anti-IL-33antibodies were tested using PBMCs from donors #603486 and #603487,while 3 anti-IL-33 antibodies were tested with PBMCs from donor #603491.All 11 of the anti-IL-33 antibodies tested on donors #603486 and #603487blocked the release of IFNγ from human PBMC induced by 260 pM IL-33,with IC₅₀ values ranging from 175 pM to 22 nM, as shown in Table 12.None of the three IL-33 antibodies tested on donor #603491 blocked therelease of IFNγ from human PBMC induced by 260 pM hIL-33 and insteadcaused an increase of IFNγ release with EC₅₀ values between 56.1 pM and189 nM.

TABLE 11 hIL-33 induced IFNγ release from human PBMC from three donors.[IL-33] Donor 603486 Donor 603487 Donor 603491 EC₅₀ (M) 1.101E−103.878E−11 2.739E−10

TABLE 12 Anti-IL-33 antibodies blocking IL-33 induced IFN-γ release fromhuman PBMC from donor #603486 and #603487 Donor #603486 Donor #603487Antibody IC₅₀ (M) IC₅₀ (M) H4H9629P 8.154E−10 5.205E−09 H4H9640P4.419E−09 1.224E−08 H4H9659P 1.252E−09 2.710E−09 H4H9660P 6.669E−102.913E−09 H4H9662P 9.640E−10 3.021E−09 H4H9663P 1.236E−08 2.203E−08H4H9664P 3.984E−09 6.081E−09 H4H9665P 1.044E−08 2.337E−08 H4H9667P8.066E−09 1.876E−08 H4H9671P 2.968E−09 8.622E−09 H4H9675P 1.754E−104.715E−10

TABLE 13 Anti-IL-33 antibodies blocking IL-33 induced IFN-γ release fromhuman PBMC from donor #603491. Donor #603491 Antibody IC₅₀ (M) H1M9559NNon-blocking H1M9566N Non-blocking H1M9568N Non-blocking

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Example 9 Human IL-33 Cross-competition Using Bio-layer Interferometry

Binding competition between a panel of different anti-IL-33 monoclonalantibodies was determined using a real time, label-free bio-layerinterferometry assay on an Octet® HTX biosensor (ForteBio, A Division ofPall Life Sciences). The experiment was performed at 25° C. using abuffer of 0.01M HEPES pH7.4, 0.15M NaCl, 0.05% v/v Surfactant Tween-20,and 0.1 mg/ml BSA (HBS-ET kinetics buffer) with the plate shaking at aspeed of 1000 rpm. To assess whether two antibodies were able to competewith one another for binding to human IL-33, a pre-mix assay format wasused where 100 nM of human IL-33 (R&D Systems; #3625-IL-010/CF) waspre-mixed with 500 nM of different anti-IL-33 monoclonal antibodies(subsequently referred to as mAb-2) for at least 2 hours prior torunning the binding competition assay. Octet biosensors coated witheither an anti-mouse Fc polyclonal antibody (Pall ForteBio Corp.,#18-5088; subsequently referred as AMC) or with an anti-human Fcpolyclonal antibody (Pall ForteBio Corp., #18-5060; subsequentlyreferred as AHC) were first submerged into wells containing 20 μg/mL ofindividual anti-IL-33 monoclonal antibodies for 3 minutes to captureanti-IL-33 monoclonal antibodies expressed either a with mouse Fc orwith a human Fc, respectively (subsequently referred to as mAb-1).Following the capture step, unoccupied anti-mouse Fc polyclonal antibodyand anti-human Fc polyclonal antibody on the Octet biosensors weresaturated by submerging them for 4 minutes into wells containing 200μg/mL of a non-specific monoclonal antibody with a mouse Fc or with ahuman Fc, respectively. Finally, the Octet biosensors were immersed for4 minutes into wells containing the pre-mixed samples of 100 nM of humanIL-33 and 500 nM of mAb-2. At the end of each cycle, the non-covalentlycaptured anti-IL-33 antibodies along with the bound pre-complex of humanIL-33 and mAb-2 were removed from the biosensors using three alternate20 second immersions into 10 mM HCl followed by submerging into HBS-ETkinetics buffer. The biosensors were washed in HBS-ET kinetics buffer inbetween every step of the experiment. The real-time binding response wasmonitored during the binding events, and the binding response (in unitsof nm) at the end of every step was recorded. During the analysis, theself-self background binding signal for a given mAb-2 (wheremAb-1=mAb-2, i.e., along the diagonal of the matrix) was subtracted fromthe observed signal for all mAb-2 binding events (across a column in thecross-competition matrix), and the background-corrected results areshown in FIG. 1. The response of mAb-1 binding to the pre-complex ofhuman IL-33 and each of the different mAb-2 samples was measured todetermine the competitive/non-competitive behavior of differentanti-IL-33 monoclonal antibodies with respect to each other.

As shown in FIG. 1 light grey boxes with black font represent bindingresponse for self-competition. Antibodies competing with each other inboth directions, independent of the order of binding, are representedwith black boxes and white font. Cells highlighted in dark grey withblack font represent the anti-IL-33 monoclonal antibody that bindsweakly to human IL-33, resulting in an observed unidirectionalcross-competition. The isotype controls used in the experiment arerepresented by dark grey boxes with white font. White boxes with blackfont represent no competition between antibodies, which suggests eachantibody has a distinct binding epitope.

Example 10 Monkey IL-33 Cross-competition Using Bio-layer Interferometry

Binding competition between a panel of different anti-IL-33 monoclonalantibodies was determined using a real time, label-free bio-layerinterferometry assay on an Octet® HTX biosensor (ForteBio, A Division ofPall Life Sciences). The experiment was performed at 25° C. using abuffer of 0.01M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v SurfactantTween-20, and 0.1 mg/ml BSA (HBS-ET kinetics buffer) with the plateshaking at a speed of 1000 rpm. To assess whether two antibodies wereable to compete with one another for binding to recombinant monkey IL-33expressed with a C-terminal hexahistidine tag (MfIL-33-6His; SEQ ID:305), approximately 0.15 nm binding units of MfIL-33-6His was firstcaptured onto anti-penta-His antibody coated Octet biosensors (FortebioInc, #18-5079) by submerging the biosensors for 85 seconds into wellscontaining 2 μg/mL of MfIL-33-6His. The antigen-captured biosensors werethen saturated with a first anti-IL-33 monoclonal antibody (subsequentlyreferred to as mAb-1) by immersion into wells containing 50 μg/mLsolution of mAb-1 for 5 minutes. The biosensors were then dipped intowells containing a 50 μg/mL solution of a second anti-IL-33 monoclonalantibody (subsequently referred to as mAb-2) for 4 minutes. Thebiosensors were washed in HBS-ET kinetics buffer in between every stepof the experiment. The real-time binding response was monitored duringthe experiment, and the maximum binding response for each binding stepwas recorded. The response of mAb-2 binding to MfIL-33-6Hispre-complexed with mAb-1 was measured, and competitive/non-competitivebehavior of different anti-IL-33 monoclonal antibodies with respect toeach other was determined.

As shown in FIG. 2, light grey boxes with black font (along a diagonal)represent self-competition (where mAb-1=mAb-2). Antibodies competing inboth directions, independent of the order of binding, are representedwith black boxes and white font. White boxes with black font representno competition between antibodies, which suggests each antibody has adistinct binding epitope. Dark grey boxes with white font represent theisotype control used in the experiment.

Example 11 mAb Testing in In Vivo Model; Acute HDM-induced LungInflammation Model to Study Role of IL-33 in Lung Inflammation

To determine the effect of an anti-IL-33 antibody, H4H9675P, in arelevant in vivo model, an acute HDM-induced lung inflammation study wasconducted in mice that were homozygous for the expression of human IL-33in place of mouse IL-33 (IL-33 HumIn mice).

IL-33 HumIn mice were intranasally administered either 50 μg of housedust mite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1×phosphate buffered saline (PBS) (n=17) or 20 μL of 1×PBS (n=3) for 5days per week for 2 weeks. A subset of the HDM challenged mice wereinjected subcutaneously with either 25 mg/kg of an anti-IL-33 antibody,H4H9675, (n=6) or an isotype control antibody (n=6) starting at threedays prior to the first HDM administration and then twice weekly untilthe end of the HDM challenge. On day 15 after the first intranasal HDM,all mice were sacrificed and their lungs were harvested. Experimentaldosing and treatment protocol for groups of mice are shown in Table 14.

TABLE 14 Experimental dosing and treatment protocol for groups of miceLength of Intranasal intranasal Group Mice challenge challenge Antibody1 IL-33 HumIn 1X PBS 2 weeks None mice 2 IL-33 HumIn 50 μg HDM in 2weeks None mice 20 μL 1X PBS 3 IL-33 HumIn 50 μg HDM in 2 weeks Isotypecontrol mice 20 μL 1X PBS 4 IL-33 HumIn 50 μg HDM in 2 weeks Anti-IL-33mice 20 μL 1X PBS antibody (H4H9675)Lung Harvest for Cytokine Analysis:

After exsanguination, the cranial and middle lobes of the right lungfrom each mouse were removed and placed into tubes containing a solutionof tissue protein extraction reagent (1× T-PER reagent; Pierce, #78510)supplemented with 1× Halt Protease inhibitor cocktail (Pierce, #78430).All further steps were performed on ice. The volume of T-PER Reagent(containing the protease inhibitor cocktail) was adjusted for eachsample to match a 1:8 (w/v) tissue to T-PER ratio. Lung samples weremanually homogenized in the tubes, using disposable pestles (KimbleChase, #749625-0010). The resulting lysates were centrifuged to pelletdebris. The supernatants containing the soluble protein extracts weretransferred to fresh tubes and stored at 4° C. until further analysis.

Total protein content in the lung protein extracts was measured using aBradford assay. For the assay, 10 μL of diluted extract samples wereplated into 96 well plates in duplicates and mixed with 200 μL of 1× DyeReagent (Biorad, #500-0006). Serial dilutions of bovine serum albumin(Sigma, #A7979), starting at 700 μg/mL in 1× T-Per reagent were used asa standard to determine the exact protein concentration of the extracts.After a 5-minute incubation at room temperature, absorbance at 595 nmwas measured on a Molecular Devices SpectraMax M5 plate reader. Dataanalysis to determine total protein content was performed using GraphPadPrism™ software.

Cytokine concentrations in the lung protein extracts were measured usinga Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScaleDiscovery, # K15048D-2), according to the manufacturer's instructions.Briefly, 50 μL/well of calibrators and samples (diluted in Diluent 41)were added to plates pre-coated with capture antibodies and incubated atroom temperature while shaking at 700 rpm for 2 hours. The plates werethen washed 3 times with 1×PBS containing 0.05% (w/v) Tween-20, followedby the addition of 25 μL of Detection Antibody Solution diluted inDiluent 45. After another 2-hour incubation at room temperature whileshaking, the plate was washed 3 times, and 150 μL of 2× Read Buffer wasadded to each well. Electrochemiluminescence was immediately read on aMSD Spector® instrument. Data analysis was performed using GraphPadPrism™ software.

Each cytokine concentration in lung total protein extracts from all micein each group was normalized to the total protein content of theextracts measured by the Bradford assay and expressed for each group asaverage pg of cytokine per mg of total lung proteins (pg/mg lungprotein, ±SD) as shown in Table 15.

Lung Harvest for Cytokine Analysis:

The level of the cytokines IL-4 and IL-5 released in the lungs of IL-33HumIn mice receiving HDM for 2 weeks was significantly higher than inIL-33 HumIn mice challenged with saline buffer. In contrast, there was atrend towards reduced IL-4 and IL-5 levels in the lungs of IL-33 HumInmice treated with anti-IL-33 antibody during the course of the acute HDMchallenge as compared to IL-33 HumIn mice administered HDM withouttreatment or with isotype control.

TABLE 15 Cytokine concentration in lung protein extracts Mean [IL-4] inMean [IL-5] in lung protein lung protein extracts (pg/mg lung extracts(pg/mg lung Experimental group protein) (±SD) protein) (±SD) 1. 1X PBSchallenge (n = 3) 0.01 (±0.01)  0.03 (±0.01) 2. HDM challenge (n = 5)1.77 (±1.63)*  4.72 (±4.14)** 3. HDM challenge + 0.79 (±0.52)*  2.03(±1.05)* Isotype control Antibody (n = 6) 4. HDM challenge + 0.30(±0.18)  0.81 (±0.67) H4H9675P (n = 6) Note: Statistical significancedetermined by Kruskal-Wallis One-way ANOVA with Dunn's multiplecomparison post-hoc test is indicated (*= p <0.05, **= p <0.01, comparedto Group 1: IL33 HumIn mice, Saline challenge).Lung Harvest for Pulmonary Cell Infiltrate Analysis

After exsanguination, the caudal lobe of the right lung from each mousewas removed, chopped into cubes that were approximately 2 to 3 mm insize, and then placed into a tube containing a solution of 20 μg/mLDNAse (Roche, #10104159001) and 0.7 U/mL Liberase TH (Roche,#05401151001) diluted in Hank's Balanced Salt Solution (HBSS) (Gibco,#14025), which was incubated in a 37° C. water bath for 20 minutes andvortexed every 5 minutes. The reaction was stopped by addingethylenediaminetetraacetic acid (Gibco, #15575) at a final concentrationof 10 mM. Each lung was subsequently dissociated using a gentleMACSDissociator® (Miltenyi Biotec, #130-095-937), then filtered through a 70μm filter and centrifuged. The resulting lung pellet was resuspended in1 mL of 1× red blood cell lysing buffer (Sigma, #R7757) to remove redblood cells. After incubation for 3 minutes at room temperature, 3 mL of1× DMEM was added to deactivate the red blood cell lysing buffer. Thecell suspensions were then centrifuged, and the resulting cell pelletswere resuspended in 5 mL of MACS buffer (autoMACS Running Buffer;Miltenyi Biotec, #130-091-221). The resuspended samples were filteredthrough a 70 μm filter and 1×10⁶ cells per well were plated in a 96-wellV-bottom plate. Cells were then centrifuged and the pellets were washedin 1×PBS. After a second centrifugation, the cell pellets wereresuspended in 100 μL of LIVE/DEAD® Fixable Aqua Dead Cell Stain (LifeTechnologies, #L34957) diluted at 1:1000 in 1× PBS to determine cellviability and incubated for 20 minutes at room temperature whileprotected from light. After one wash in 1×PBS, cells were incubated in asolution of MACS buffer containing 10 μg/mL of purified rat anti-mouseCD16/CD32 Fc Block, (Clone: 2.4G2; BD Biosciences, #553142) for 10minutes at 4° C. The cells were washed once and then incubated in theappropriate antibody mixture (described in Table 16) diluted in MACSbuffer for 30 minutes at 4° C. while protected from light. Afterantibody incubation, the cells were washed twice in MACS buffer,resuspended in BD cytofix (BD Biosciences, #554655) and then incubatedfor 15 minutes at 4° C. while protected from light. The cells weresubsequently washed, resuspended in MACS buffer, and then transferred toBD FACS tubes (BD Biosciences, #352235) for analysis of eosinophils,innate lymphoid cell type 2 (ILC2) and lymphocytes by flow cytometry.

Activated CD4 T cells were defined as cells that were live, CD45⁺,SSC^(Lo), FSC^(Lo), CD3⁺, CD19⁻, CD4⁺, CD8⁻, and CD69⁺. Activated Bcells were defined as cells that were live, CD45⁺, SSC^(Lo), FSC^(Lo),CD3⁻, CD19⁺, and CD69⁺. Eosinophils were defined as live, CD45⁺, GR1⁻,CD11c^(lo), SiglecF^(hi). ILC2 cells were defined as live, CD45⁺,Lineage-(Lineage: CD19, CD3, CD11 b, CD11c, F4/80), CD127+, Sca⁻1⁺,ST2⁺. Data for activated CD4 cells, expressed as frequency of activatedcells (CD69⁺) within the parent population (CD4, ±SD), are shown inTable 17.

TABLE 16 Antibodies Used for Flow Cytometry Analysis Catalog FinalAntibody Fluorochrome Manufacturer Number dilution CD11c APC BDBiosciences 550261 1/100 CD45 PerCP Cy5.5 eBiosciences 45-0454-82 1/800F4/80 Pacific Blue eBiosciences 48-4801-82 1/200 Siglec-F PE BDBiosciences 552126 1/100 Ly6G (Gr-1) APC-eFluor780 eBiosciences47-5931-82 1/200 CD3 PE-Cy7 BD Biosciences 552774 1/200 CD19 eFluor 450eBiosciences 48-0193-82 1/200 CD4 APC-H7 BD Biosciences 560181 1/200 CD8APC eBiosciences 17-0081-82 1/200 CD69 PE eBiosciences 12-0691-82 1/200CD3 eFluor 450 eBiosciences 48-0031-82 1/200 CD11b eFluor450eBiosciences 40-0112-82 1/100 CD11c eFluor450 eBiosciences 48-0114-821/100 CD127 APC-eFluor780 eBiosciences 47-1271-82 1/200 Sca-1 FITC BDBiosciences 557405 1/200 ST2 APC Biolegend 145306 1/200Pulmonary Cell Infiltrate Analysis:

As shown in Table 17, the frequency of activated CD4⁺ T cells,eosinophils, and ILC2 in the lungs of IL-33 HumIn mice receiving HDM for2 weeks was significantly higher than in IL-33 HumIn mice challengedwith 1×PBS control. In contrast, a trend towards a reduced frequency ofthese infiltrates was observed in IL-33 HumIn mice when treated with theanti-IL-33 antibody during the course of the acute HDM challenge ascompared to IL-33 HumIn mice administered HDM without treatment or withisotype control.

A trend towards an increase in the frequency of activated B cells wasobserved in the lungs of IL33 HumIn mice challenged with HDM for 2 weekscompared to IL33 HumIn mice challenged with 1×PBS control. Uponanti-IL-33 antibody treatment, a significant reduction in the frequencyof pulmonary activated B cells in the lungs of IL33 HumIn micechallenged with HDM was observed, as compared to IL-33 HumIn miceadministered HDM without treatment or with isotype control.

TABLE 17 Frequency of pulmonary cell infiltrate as determined by flowcytometry Mean Frequency of Mean Mean Mean activated Frequency ofFrequency of Frequency of CD4+ T cells activated B eosinophils in ILC2in in CD4+ cells in the B CD45+ Lymphoid population cell populationpopulation population Experimental group (±SD) (±SD) (±SD) (±SD) 1. 1XPBS challenge 6.17 (±0.59)  6.85 (±3.09) 2.55 (±0.79) 0.33 (±0.05)  (n =3) 2. HDM challenge 29.52 (±8.57)* 10.13 (±3.30) 17.28 (±3.97)* 1.15(±0.37)* (n = 5) 3. HDM challenge + 29.68 (±9.84)* 11.01 (±2.31)  19.19(±11.55)* 1.57 (±0.78)* Isotype control Antibody (n = 6) 4. HDMchallenge + 16.38 (±3.30)    4.88 (±1.70)^(†) 10.32 (±4.63)  0.53(±0.12)  H4H9675P (n = 6) Note: Statistical significance determined byKruskal-Wallis One-way ANOVA with Dunn's multiple comparison post-hoctest is indicated (*= p <0.05, **= p <0.01, compared to groups 1: IL33HumIn mice, Saline challenge; ^(†)p <0.05, compared to group 3: IL33Humin mice, HDM challenge 2 weeks + Isotype control antibody).

Example 12 mAb Testing in In Vivo Model; Chronic HDM-induced Fibrosisand Severe Lung Inflammation Model to Study Role of IL-33 in LungInflammation

To determine the effect of an anti-IL-33 antibody, H4H9675P, in arelevant in vivo model, a chronic HDM-induced fibrosis and severe lunginflammation study was conducted in mice that were homozygous for theexpression of human IL-33 in place of mouse IL-33 (IL-33 HumIn mice).

IL-33 HumIn mice were intranasally administered either 50 μg house dustmite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1× phosphatebuffered saline (PBS) or 20 μL of 1×PBS for 5 days per week for 12weeks. A second control group of IL33 HumIn mice were administered 50 μgHDM extract diluted in 20 μL of 1×PBS for 5 days per week for 4 weeks,to assess the severity of the disease at the onset of antibodytreatment. Two groups of HDM challenged mice were injectedsubcutaneously with 25 mg/kg of either an anti-IL-33 antibody, H4H9675P,or an isotype control antibody starting after 4 weeks of HDM challengeand then twice per week until the end of the HDM challenge (8 weeks ofantibody treatment). On day 85 of the study, all mice were sacrificedand their lungs were harvested. Experimental dosing and treatmentprotocol for groups of mice are shown in Table 18.

TABLE 18 Experimental dosing and treatment protocol for groups of miceLength of Intranasal intranasal Group Mice challenge challenge Antibody1 IL-33 HumIn 1X PBS 12 weeks None mice 2 IL-33 HumIn 50 μg HDM in  4weeks None mice 20 μL 1X PBS 3 IL-33 HumIn 50 μg HDM in 12 weeks Nonemice 20 μL 1X PBS 4 IL-33 HumIn 50 μg HDM in 12 weeks Isotype controlmice 20 μL 1X PBS antibody 5 IL-33 HumIn 50 μg HDM in 12 weeksAnti-IL-33 antibody mice 20 μL 1X PBS (H4H9675P)Lung Harvest for Cytokine Analysis:

After exsanguination, the cranial and middle lobes of the right lungfrom each mouse were removed and placed into tubes containing a solutionof tissue protein extraction reagent (1× T-PER reagent; Pierce, #78510)supplemented with 1× Halt Protease inhibitor cocktail (Pierce, #78430).All further steps were performed on ice. The volume of T-PER Reagent(containing the protease inhibitor cocktail) was adjusted for eachsample to match a 1:8 (w/v) tissue to T-PER ratio. Lung samples weremanually homogenized in the tubes, using disposable pestles (KimbleChase, #749625-0010). The resulting lysates were centrifuged to pelletdebris. The supernatants containing the soluble protein extracts weretransferred to fresh tubes and stored at 4° C. until further analysis.

Total protein content in the lung protein extracts was measured using aBradford assay. For the assay, 10 μL of diluted extract samples wereplated into 96 well plates in duplicates and mixed with 200 μL of 1× DyeReagent (Biorad, #500-0006). Serial dilutions of bovine serum albumin(BSA; Sigma, #A7979), starting at 700 μg/mL in 1× T-Per reagent wereused as a standard to determine the protein concentration of theextracts. After a 5-minute incubation at room temperature, absorbance at595 nm was measured on a Molecular Devices SpectraMax M5 plate reader.Data analysis to determine total lung extract protein content based onthe BSA standard was performed using GraphPad Prism™ software.

Cytokine concentrations in the lung protein extracts were measured usinga Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScaleDiscovery, # K15048D-2), according to the manufacturer's instructions.Briefly, 50 μL/well of calibrators and samples (diluted in Diluent 41)were added to plates pre-coated with capture antibodies and incubated atroom temperature while shaking at 700 rpm for 2 hours. The plates werethen washed 3 times with 1×PBS containing 0.05% (w/v) Tween-20, followedby the addition of 25 μL of Detection Antibody Solution diluted inDiluent 45. After another 2 hour incubation at room temperature whileshaking, the plate was washed 3 times, and 150 μL of 2× Read Buffer wasadded to each well. Electrochemiluminescence was immediately read on aMSD Spector® instrument. Data analysis was performed using GraphPadPrism software.

Each cytokine concentration in lung total protein extracts from all micein each group was normalized to the total protein content of theextracts measured by the Bradford assay, and expressed for each group asaverage pg of cytokine per mg of total lung proteins (pg/mg lungprotein, ±SD) as shown in Table 19.

TABLE 19 Cytokine concentration in lung protein extracts Mean [IL-4] inMean [IL-5] in lung protein lung protein extracts (pg/mg lung extracts(pg/mg lung Experimental group protein) (±SD) protein) (±SD) 1. 1X PBSchallenge, 12 0.03 (±0.01) 0.08 (±0.05) weeks (n = 5) 2. HDM challenge,4 weeks  2.84 (±2.22)*  4.44 (±4.00)** (n = 6) 3. HDM challenge, 12 7.31 (±3.94)**  6.23 (±3.81)* weeks (n = 3) 4. HDM challenge, 12 2.28(±1.94) 3.39 (±3.29) weeks + Isotype control antibody (n = 2) 5. HDMchallenge, 12 0.38 (±0.21) 0.48 (±0.17) weeks + H4H9675P (n = 5) Note:Statistical significance determined by Kruskal-Wallis One-way ANOVA withDunn's multiple comparison post-hoc test is indicated (*= p <0.05, **= p<0.01, compared to groups 1: IL33 HumIn mice, Saline challenge).Lung Cytokines Analysis:

The level of the cytokines IL-4 and IL-5 released in the lungs of IL-33HumIn mice receiving HDM for 4 and 12 weeks was significantly higherthan in IL-33 HumIn mice challenged with 1×PBS. In contrast, there was atrend towards reduced IL-4 and IL-5 levels in the lungs of IL-33 HumInmice treated with anti-IL-33 antibody during the course of the chronicHDM challenge as compared to IL-33 HumIn mice administered HDM withouttreatment or with isotype control.

What is claimed is:
 1. A method for treating an inflammatory disease ordisorder of the respiratory tract or the lungs, or both, or at least onesymptom associated with the inflammatory disease or disorder, the methodcomprising administering an antibody that binds specifically to humaninterleukin-33 (IL-33), or an antigen-binding fragment thereof, or apharmaceutical composition containing an effective amount of an antibodythat binds specifically to IL-33, or an antigen-binding fragmentthereof, to a patient in need thereof, wherein the inflammatory diseaseor disorder is alleviated, or reduced in severity, duration or frequencyof occurrence, or at least one symptom associated with the inflammatorydisease or disorder is alleviated, or reduced in severity, duration, orfrequency of occurrence, and wherein the antibody or antigen-bindingfragment thereof is an isolated human monoclonal antibody orantigen-binding fragment thereof comprising: (a) the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR)comprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 50, 82, 98, 130, 178, and 274; and (b) the CDRs of a lightchain variable region (LCVR) comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 58, 90, 106, 138, 186, and 282.2. The method of claim 1, wherein the inflammatory disease or disorderis selected from the group consisting of asthma, chronic obstructivepulmonary disease (COPD), and allergic rhinitis.
 3. The method of claim2, wherein the inflammatory disease or disorder is asthma.
 4. The methodof claim 3, wherein the asthma is eosinophilic or non-eosinophilicasthma.
 5. The method of claim 3, wherein the asthma is steroidresistant or steroid sensitive asthma.
 6. The method of claim 2, whereinthe inflammatory disease or disorder is chronic obstructive pulmonarydisease (COPD).
 7. The method of claim 6, wherein the chronicobstructive pulmonary disease results from, or is caused in part bycigarette smoke.
 8. The method of claim 1, further comprisingadministering an effective amount of a second therapeutic agent usefulfor alleviating the inflammatory disease or disorder, or at least onesymptom of the inflammatory disease or disorder.
 9. The method of claim8, wherein the second therapeutic agent is selected from the groupconsisting of a non-steroidal anti-inflammatory drug (NSAID), acorticosteroid, a bronchial dilator, an antihistamine, epinephrine, adecongestant, a thymic stromal lymphopoietin (TSLP) antagonist, an IL-13antagonist, an IL-4 antagonist, an IL-4/IL-13 dual antagonist, an IL-5antagonist, an IL-6 antagonist, an IL-12/23 antagonist, an IL-22antagonist, an IL-25 antagonist, an IL-17 antagonist, an IL-31antagonist, an oral PDE4 inhibitor and another IL-33 antagonist or adifferent antibody to IL-33.
 10. The method of claim 1, wherein theantibody or antigen-binding fragment comprises the heavy and light chainCDRs of a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: SEQ ID NOs: 50/58, 82/90, 98/106, 130/138, 178/186, and274/282.
 11. The method of claim 1, wherein the antibody orantigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, selected from the group consisting of: SEQ IDNOs: 52-54-56-60-62-64; 84-86-88-92-94-96; 100-102-104-108-110-112;132-134-136-140-142-144; 180-182-184-188-190-192; and276-278-280-284-286-288.
 12. The method of claim 1, wherein the antibodyor antigen-binding fragment comprises: (a) a heavy chain variable region(HCVR) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 50, 82, 98, 130, 178, and 274; and (b) a lightchain variable region (LCVR) comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 58, 90, 106, 138, 186, and 282.13. The method of claim 12, wherein the antibody or antigen-bindingfragment comprises a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of: SEQ ID NOs: 50/58, 82/90, 98/106, 130/138,178/186, and 274/282.
 14. The method of claim 13, wherein the antibodyor antigen-binding fragment comprises a HCVR/LCVR amino acid sequencepair of SEQ ID NOs: 50/58.
 15. The method of claim 13, wherein theantibody or antigen-binding fragment comprises a HCVR/LCVR amino acidsequence pair of SEQ ID NOs: 82/90.
 16. The method of claim 13, whereinthe antibody or antigen-binding fragment comprises a HCVR/LCVR aminoacid sequence pair of SEQ ID NOs: 98/106.
 17. The method of claim 13,wherein the antibody or antigen-binding fragment comprises a HCVR/LCVRamino acid sequence pair of SEQ ID NOs: 130/138.
 18. The method of claim13, wherein the antibody or antigen-binding fragment comprises aHCVR/LCVR amino acid sequence pair of SEQ ID NOs: 178/186.
 19. Themethod of claim 13, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.20. The method of claim 3, wherein the asthma comprises an asthmaexacerbation.
 21. A method for treating a patient who demonstrates asensitivity to an allergen, the method comprising administering aneffective amount of an antibody or antigen binding fragment thereof thatbinds specifically to IL-33, or a pharmaceutical composition containingan effective amount of an antibody or antigen binding fragment thereofthat binds specifically to IL-33, to a patient in need thereof, whereinthe patient demonstrates a reduced sensitivity to, or a diminishedallergic reaction against the allergen, or does not experience anysensitivity or allergic reaction to, or anaphylactic response to theallergen following administration of the antibody or a compositioncomprising the antibody, and wherein the antibody or antigen-bindingfragment thereof is an isolated human monoclonal antibody orantigen-binding fragment thereof comprising: (a) the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR)comprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 50, 82, 98, 130, 178, and 274; and (b) the CDRs of a lightchain variable region (LCVR) comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 58, 90, 106, 138, 186, and 282.22. The method of claim 21, further comprising administering aneffective amount of a second therapeutic agent useful for diminishing anallergic response to an allergen, or at least one symptom of theallergic response.
 23. The method of claim 22, wherein the secondtherapeutic agent is selected from the group consisting of anon-steroidal anti-inflammatory drug (NSAID), a corticosteroid, abronchial dilator, an antihistamine, epinephrine, a decongestant, athymic stromal lymphopoietin (TSLP) antagonist, an IL-13 antagonist, anIL-4 antagonist, an IL-4/IL-13 dual antagonist, an IL-5 antagonist, anIL-6 antagonist, an IL-12/23 antagonist, an IL-22 antagonist, an IL-25antagonist, an IL-17 antagonist, an IL-31 antagonist, an oral PDE4inhibitor and another IL-33 antagonist or a different antibody to IL-33.24. The method of claim 21, wherein the antibody or antigen-bindingfragment comprises the heavy and light chain CDRs of a HCVR/LCVR aminoacid sequence pair selected from the group consisting of: SEQ ID NOs:50/58, 82/90, 98/106, 130/138, 178/186, and 274/282.
 25. The method ofclaim 21, wherein the antibody or antigen-binding fragment comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected fromthe group consisting of: SEQ ID NOs:52-54-56-60-62-64;84-86-88-92-94-96; 100-102-104-108-110-112; 132-134-136-140-142-144;180-182-184-188-190-192; and 276-278-280-284-286-288.
 26. The method ofclaim 21, wherein the antibody or antigen-binding fragment comprises:(a) a heavy chain variable region (HCVR) comprising the amino acidsequence selected from the group consisting of SEQ ID NOs: 50, 82, 98,130, 178, and 274; and (b) a light chain variable region (LCVR)comprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 58, 90, 106, 138, 186, and
 282. 27. The method of claim 26,wherein the antibody or antigen-binding fragment comprises a HCVR/LCVRamino acid sequence pair selected from the group consisting of: SEQ IDNOs: 50/58, 82/90, 98/106, 130/138, 178/186, and 274/282.
 28. The methodof claim 27, wherein the antibody or antigen-binding fragment comprisesa HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 50/58.
 29. Themethod of claim 27, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 82/90. 30.The method of claim 27, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 98/106.31. The method of claim 27, wherein the antibody or antigen-bindingfragment comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs:130/138.
 32. The method of claim 27, wherein the antibody orantigen-binding fragment comprises a HCVR/LCVR amino acid sequence pairof SEQ ID NOs: 178/186.
 33. The method of claim 27, wherein the antibodyor antigen-binding fragment comprises a HCVR/LCVR amino acid sequencepair of SEQ ID NOs: 274/282.
 34. A method for treating an inflammatorydisease or disorder of the respiratory tract or the lungs, or both or atleast one symptom associated with the inflammatory disease or disorder,the method comprising administering an antibody that binds specificallyto IL-33, or an antigen-binding fragment thereof, or a pharmaceuticalcomposition containing an effective amount of an antibody that bindsspecifically to IL-33, or an antigen-binding fragment thereof, to apatient in need thereof, wherein the inflammatory disease or disorder isalleviated, or reduced in severity, duration or frequency of occurrence,or at least one symptom associated with the inflammatory disease ordisorder is alleviated, or reduced in severity, duration, or frequencyof occurrence, and wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region (HCVR) comprising theamino acid sequence selected from the group consisting of SEQ ID NOs:50, 82, 98, 130, 178, and
 274. 35. The method of claim 34, wherein theinflammatory disease or disorder is asthma.
 36. The method of claim 34,wherein the inflammatory disease or disorder is chronic obstructivepulmonary disease (COPD).
 37. A method for treating a patient whodemonstrates a sensitivity to an allergen, the method comprisingadministering an effective amount of an antibody or antigen bindingfragment thereof that binds specifically to IL-33, or a pharmaceuticalcomposition containing an effective amount of an antibody or antigenbinding fragment thereof that binds specifically to IL-33, to a patientin need thereof, wherein the patient demonstrates a reduced sensitivityto, or a diminished allergic reaction against the allergen, or does notexperience any sensitivity or allergic reaction to, or anaphylacticresponse to the allergen following administration of the antibody or acomposition comprising the antibody, and wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable region(HCVR) comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 50, 82, 98, 130, 178, and
 274. 38. A methodfor treating an inflammatory disease or disorder of the respiratorytract or the lungs, or both, the method comprising administering to asubject in need thereof an antibody that binds specifically to humanIL-33, or an antigen-binding fragment thereof, wherein the antibody orantigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, of SEQ ID NOs: 276-278-280-284-286-288.